Anti-cancer compositions and methods

ABSTRACT

Anti-cancer compositions and methods are described including one or more isothiocyanates and/or isoselenocyanates. Methods of treating a subject are provided according to embodiments of the present invention which include administering a therapeutically effective amount of a composition including an isothiocyanate and/or isoselenocyanate to a subject having a condition characterized by Akt dysregulation. Administering a therapeutically effective amount of a composition including an isothiocyanate and/or isoselenocyanate to a subject detectably increases apoptosis and/or decreases proliferation of cancer cells, particularly cancer cells characterized by Akt dysregulation.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/102,629, filed Apr. 14, 2008, which claims priority from U.S.Provisional Patent Application Ser. Nos. 60/911,565, filed Apr. 13, 2007and 60/959,554, filed Jul. 13, 2007, the entire content of all of whichis incorporated herein by reference.

GOVERNMENT SPONSORSHIP

The invention was made with government support under Grant Nos.CB056603, CA127892, CA136667, CA128033 and HHSN261200566003C awarded byThe National Institutes of Health. The Government has certain rights inthe invention.

FIELD OF THE INVENTION

The present invention relates generally to anti-cancer compositions andmethods. In specific embodiments, the present invention relates tocompositions including one or more isothiocyanates and/orisoselenocyanates, methods for treatment and/or prevention ofpathological conditions in a subject using one or more isothiocyanatesand/or isoselenocyanates and methods for synthesis of particularisoselenocyanates.

BACKGROUND OF THE INVENTION

In spite of recent medical progress, cancer continues to be one of themost common and deadly diseases. Elucidation of biochemical pathwaysinvolved in development and progression of various cancers is importantto identify potential anti-cancer treatments as well as to developagents effective to regulate such pathways in other aspects of healthand disease.

A particular cancer, melanoma, is the most deadly form of skin cancerdue to its high metastatic potential. Akt3 and downstream PRAS40 arepart of a key signaling cascade activated in ˜70% of melanomas. Akt3functions to reduce cellular apoptosis in early melanomas, therebypromoting development of this disease. Compositions and methods arerequired to inhibit the Akt pathway and inhibit abnormal cell survivaland proliferation.

SUMMARY OF THE INVENTION

Compositions including an isoselenocyanate having the structural formulaR—(CH₂)_(n)—N═C═Se, where n is an integer in the range of 1-8,inclusive, and where R is selected from the group consisting of: anaromatic group and a non-aromatic organic group are described herein.

In embodiments of the present invention, a composition including anisoselenocyanate having the structural formula:

where R′ is a substituted or unsubstituted, branched or straight chain,lower alkyl group, and where n is an integer in the range of 3-8,inclusive is provided along with methods of synthesis and use of suchcompositions. In particular embodiments, R′ is CH₃.

Embodiments of compositions of the present invention include anisoselenocyanate having the structural formula:

where R′ is a substituted or unsubstituted, branched or straight chain,lower alkyl group, and where n is an integer in the range of 3-8,inclusive. In particular embodiments, R′ is CH₃ and n is 4.

Methods for synthesis of an isoselenocyanate having the structuralformula: R—(CH₂)_(n)—N═C═Se, where n is an integer in the range of 1-8,inclusive, and where R is selected from the group consisting of: anunsubstituted aromatic group; an aromatic group substituted by one ormore substituents selected from the group consisting of: F, Cl, Br, alower alkyl group, a lower alkoxy group and a fluorinated lower alkylgroup; R′—S(O), where R′ is a lower alkyl group, which may besubstituted or unsubstituted, branched or straight chain; and CH₂═CH,are provided according to the present invention. Methods of synthesis ofthe present invention include formylation of a starting alkylaminecompound having the structural formula: R—(CH₂)_(n)—NH₂, where n is aninteger in the range of 1-8, inclusive, where R is selected from thegroup consisting of: an unsubstituted aromatic group; an aromatic groupsubstituted by one or more substituents selected from the groupconsisting of: F, Cl, Br, a lower alkyl group, a lower alkoxy group anda fluorinated lower alkyl group; R′—S(O), where R′ is a lower alkylgroup, which may be substituted or unsubstituted, branched or straightchain; and CH₂═CH, to produce a formylated intermediate having thestructural formula: R—(CH₂)_(n)—NHCHO, where R and n are identical to Rand n of the starting alkylamine; and contacting the formylatedintermediate with triphosgene and selenium powder in the presence oftriethylamine. In a preferred option, the formylated intermediate iscontacted with triphosgene and selenium powder in the presence oftriethylamine and in the presence of a solvent, such as dichloromethane.

Pharmaceutical compositions are provided according to embodiments of thepresent invention which include one or more isothiocyanates and/orisoselenocyanates having the structural formula R—(CH₂)_(n)—N═C═X, wheren is an integer in the range of 1-8, inclusive, where X is S or Se, andwhere R is selected from the group consisting of: an aromatic group anda non-aromatic organic group.

Pharmaceutical compositions according to embodiments of the presentinvention include one or more isothiocyanates and/or isoselenocyanateshaving the structural formula phenyl-(CH₂)_(n)—N═C═X, where n is aninteger in the range of 1-8, inclusive, and where X is S or Se.

Pharmaceutical compositions are provided according to embodiments of thepresent invention which include an isoselenocyanate having thestructural formula selected from the group consisting of:

where n is 4 or 6;

and

where R′ is a substituted or unsubstituted, branched or straight chain,lower alkyl group, and where n is an integer in the range of 3-8,inclusive; and a pharmaceutically acceptable carrier. Optionally, thepharmaceutically acceptable carrier is a particulate carrier. In afurther option, the pharmaceutical composition is formulated for topicalapplication.

Methods of treating a subject are provided according to the presentinvention which include administering an effective amount of acomposition including an isoselenocyanate described herein to a subjectin need thereof. In embodiments of the present invention, a compositionadministered to a subject in need thereof includes a phenylalkylisoselenocyanate having the structural formula:

where n is 4 or 6. Optionally, the phenylalkyl moiety is substituted atan available substitutable site. For example, the phenylalkyl may besubstituted by one or more substituents selected from the groupconsisting of: F, Cl, Br, a lower alkyl group, a lower alkoxy group anda fluorinated lower alkyl group.

Embodiments of compositions administered to a subject in need thereofinclude an isoselenocyanate having the structural formula:

where R′ is a substituted or unsubstituted, branched or straight chain,lower alkyl group, and where n is an integer in the range of 3-8,inclusive. Optionally, R′ is CH₃. In a further option, R′ is CH₃ and nis 4.

An isoselenocyanate composition is optionally conjugated to glutathione,cysteine or N-acetylcysteine to produce an isoselenocyanate glutathioneconjugate; an isoselenocyanate cysteine conjugate; and anisoselenocyanate N-acetylcysteine conjugate for administration to asubject in need thereof.

In embodiments of methods including administration of anisoselenocyanate to a subject, the subject is human.

In further embodiments, the subject has or is at risk of having cancer.In certain embodiments, the subject has cancer or is at risk for cancercharacterized by dysregulation of Akt1, Akt2 and/or Akt3. In furtherembodiments, the cancer is a melanoma.

Methods of treating a subject are provided according to embodiments ofthe present invention which include administering a therapeuticallyeffective amount of a composition including an isothiocyanate and/orisoselenocyanate to a subject in need thereof, wherein theisothiocyanate or isoselenocyanate has the structural formula:R—(CH₂)_(n)—N═C═X, where n is an integer in the range of 1-8, inclusive,where X is S or Se, and where R is selected from the group consistingof: an aromatic group and a non-aromatic organic group and wherein thesubject has a condition characterized by Akt dysregulation, such ascancer, pre-neoplastic hyperproliferation, cancer in-situ, neoplasms,metastasis, tumor or benign growth. In certain embodiments of methods oftreatment of a subject, contacting cells characterized by Aktdysregulation with a therapeutic amount of an isothiocyanate and/orisoselenocyanate described herein decreases a component of an Aktsignaling pathway selected from the group consisting of: an Akt1signaling pathway; an Akt2 signaling pathway; an Akt3 signaling pathway;and a combination thereof. For example, contacting the cell with anisothiocyanate and/or isoselenocyanate decreases a component of an Aktsignaling pathway selected from pAkt1, pAkt2, pAk3, pPRAS40 and acombination thereof. In embodiments of described methods, treatment of asubject with a therapeutically effective amount of the compositionincluding an isothiocyanate and/or isoselenocyanate is substantiallywithout toxic effect on cells in which Akt is not dysregulated.

Methods of treating a subject are provided according to embodiments ofthe present invention which include administering a therapeuticallyeffective amount of a composition including an isothiocyanate and/orisoselenocyanate to a subject in need thereof, wherein theisothiocyanate or isoselenocyanate has the structural formula:phenyl-(CH₂)_(n)—N═C═X, where n is an integer in the range of 1-8,inclusive, where X is S or Se, and wherein the subject has a conditioncharacterized by Akt dysregulation, such as cancer, pre-neoplastichyperproliferation, cancer in-situ, neoplasms, metastasis, tumor orbenign growth. Optionally the phenyl group is substituted.

Methods according to embodiments of the present invention includeadministering a therapeutically effective amount of a compositionincluding an isoselenocyanate and/or an isothiocyanate to a subjectwherein the administration detectably increases apoptosis and/ordecreases proliferation of cells of the cancer, pre-neoplastichyperproliferation, cancer in-situ, neoplasms, metastasis, tumor orbenign growth.

Optionally, a composition including an isoselenocyanate and/orisothiocyanate according to embodiments of the present invention isformulated for topical application, for instance to treat cancer,pre-neoplastic hyperproliferation, cancer in-situ, neoplasms,metastasis, tumor or benign growth of the skin.

Optionally, methods of the present invention additionally includeadministration of an adjunct anti-cancer treatment.

A method of modulating Akt dysregulation in a cell is provided accordingto embodiments of the present invention which includes contacting thecell with an effective amount of an isothiocyanate or isoselenocyanatehaving the structural formula: R—(CH₂)_(n)—N═C═X, where n is an integerin the range of 1-8, inclusive, where X is S or Se, and where R isselected from the group consisting of: an aromatic group and anon-aromatic organic group. In certain embodiments of methods ofmodulating Akt dysregulation, contacting the cell with anisoselenocyanate decreases a component of an Akt signaling pathwayselected from the group consisting of: an Akt1 signaling pathway; anAkt2 signaling pathway; an Akt3 signaling pathway; and a combinationthereof. For example, contacting the cell with an isoselenocyanatedecreases a component of an Akt signaling pathway selected from pAkt1,pAkt2, pAk3, pPRAS40 and a combination thereof.

A method of modulating Akt dysregulation in a cell is provided accordingto embodiments of the present invention which includes contacting thecell with an effective amount of an isothiocyanate or isoselenocyanatehaving the structural formula: phenyl-(CH₂)_(n)—N═C═X, where n is aninteger in the range of 1-8, inclusive, and where X is S or Se.Optionally, the phenyl group is substituted.

A method of modulating Akt dysregulation in a cell is provided accordingto embodiments of the present invention which includes contacting thecell with an effective amount of an isoselenocyanate having thestructural formula: R—(CH₂)_(n)—N═C═Se, where n is an integer in therange of 1-8, inclusive, and where R is selected from the groupconsisting of: an aromatic group and a non-aromatic organic group. Incertain embodiments of methods of modulating Akt dysregulation,contacting the cell with an isoselenocyanate decreases a component of anAkt signaling pathway selected from the group consisting of: an Akt1signaling pathway; an Akt2 signaling pathway; an Akt3 signaling pathway;and a combination thereof. For example, contacting the cell with anisoselenocyanate decreases a component of an Akt signaling pathwayselected from pAkt1, pAkt2, pAk3, pPRAS40 and a combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a bar graph showing a comparison of cell viability followingexposure to PBITC or ISC-4, compared to controls;

FIG. 1B is a bar graph showing a comparison of cell viability followingexposure to PHITC or ISC-6, compared to controls;

FIG. 2A is a bar graph of results of proliferation analysis of UACC 903cells treated with DMSO or different concentrations (5 uM, 10 uM, 15 uM)of API-2, PHSC, PBITC or ISC-4 for 24 hours;

FIG. 2B is a bar graph of results of proliferation analysis of UACC 903cells treated with DMSO or different concentrations (5 uM, 10 uM, 15 uM)of API-2, PHSC, PHITC or ISC-6 for 24 hours;

FIG. 3A is a bar graph showing the effects of treatment of UACC 903cells in culture with DMSO or 5 μM, 10 μM or 15 μM of API-2, PHSC, PBITCor ISC-4 for 24 hours on caspase-3/7 activity, an indicator ofapoptosis;

FIG. 3B is a bar graph showing the effects of treatment of UACC 903cells in culture with DMSO or 5 μM, 10 μM or 15 μM of API-2, PHSC, PHITCor ISC-6 for 24 hours on caspase-3/7 activity, an indicator ofapoptosis;

FIG. 4A is a bar graph showing results of cell cycle analysis of UACC903 cells treated with controls (API-2, PHSC), PBITC or ISC-4;

FIG. 4B is a bar graph showing results of cell cycle analysis of UACC903 cells treated with controls (API-2, PHSC), PHITC or ISC-6;

FIG. 5A is a bar graph showing the effect of isothiocyanates on tumordevelopment;

FIG. 5B is a bar graph showing the effect of isoselenocyanates on tumordevelopment;

FIG. 6A is a line graph showing change in tumor size and body weight(inset) over time in mice treated i. p. with PBITC or PHITC (0.76μmoles) or ISC-4 or ISC-6 (0.76 μmoles, equivalent to 3 ppm selenium);

FIG. 6B is a line graph showing change in tumor size and body weight(inset) over time in mice treated i. p. with PBITC or PHITC (0.76μmoles) or ISC-4 or ISC-6 (0.76 μmoles, equivalent to 3 ppm selenium);

FIG. 7A is a bar graph showing results of analysis of apoptosisfollowing ISC-4 treatment compared to treatment with PBITC or DMSO, insize and time matched tumors;

FIG. 7B is a bar graph showing results of analysis of cell proliferationfollowing ISC-4 treatment compared to treatment with PBITC or DMSO, insize and time matched tumors;

FIG. 8 is a bar graph showing results of analysis of levels of SGOT,SGPT, alkaline phosphatase, glucose and creatinine in blood collectedfrom animals treated with PBITC, ISC-4 or DMSO vehicle;

FIG. 9 is a reproduction of an image of an immunoblot showing dosedependent decreases in phosphorylated (active) Akt (S473) and downstreamPRAS40 (T246) and a corresponding dose dependent increase in cleavedPARP, reflective of high levels of cellular apoptosis in cells treatedwith ISC-4 or ISC-6;

FIG. 10A is a reproduction of an image of an immunoblot showing theeffect of ISC-4 on Akt signaling pathway in melanoma cell line 1205 Lu;

FIG. 10B is a reproduction of an image of an immunoblot showing theeffect of ISC-4 on Akt signaling pathway in melanoma cell line WM115;

FIG. 11 is a bar graph showing quantitation of immunoblot analysis oftumor protein lysates from animals treated with DMSO, PBITC or ISC-4 andindicates decreased relative expression of phosphorylated (active) Aktand downstream PRAS40 of Akt3;

FIG. 12 is a line graph showing that decreased expression (activity) ofAkt3 reduced the tumor size in animals injected with Akt3 siRNA treatedcells compared to control cells nucleofected with scrambled siRNA ornucleofection buffer.

FIG. 13 is a reproduction of an image of an immunoblot analysis of tumorprotein lysates showing reduction in expression of Akt3 in tumors bysiRNA directed against Akt3;

FIG. 14A is a line graph showing inhibition of tumor development by1-isoselenocyanto-4-methylsulfinylbutane (SFN Iso Se, alsointerchangeably called ISC-SFN4 herein) compared to controls.

FIG. 14B is a line graph showing a lack of significant changes in bodyweight in treated animals, indicating that a lack of toxicity of theadministered compounds;

FIG. 15 is a bar graph showing results of a cell viability assaymeasuring inhibitory efficacy of DMSO, ISC-1, ISC-2, ISC-4, ISC-6, BITC,PEITC, PBITC, or PHITC on UACC 903 cells;

FIG. 16 is a bar graph showing the effect of DMSO or 5-15 μM of ISC-1,ISC-2, ISC-4, ISC-6, BITC, PEITC, PBITC, or PHITC on cell proliferation;

FIG. 17 is a bar graph showing the effect of DMSO or 5-15 μM of ISC-1,ISC-2, ISC-4, ISC-6, BITC, PEITC, PBITC, or PHITC on apoptosis;

FIG. 18A is a line graph showing effect of selected isoselenocyanates ontumor size;

FIG. 18B is a line graph showing effect of selected isoselenocyanates onbody weight;

FIG. 18C is a line graph showing effect of selected isothiocyanates ontumor size; and

FIG. 18D is a line graph showing effect of selected isothiocyanates onbody weight;

FIG. 19 is a bar graph showing the effects of topically applied PBITC orISC-4 on reconstructed human skin containing GFP tagged UACC 903 humanmelanoma cells;

FIG. 20 is a pair of line graphs showing the effect of topical ISC-4application on melanoma tumor growth in vivo; and

FIG. 21 is a pair of line graphs showing the effect of topical ISC-4application on melanoma tumor growth in vivo.

DETAILED DESCRIPTION OF THE INVENTION

Anti-cancer compositions and methods are provided according toembodiments of the present invention. In certain embodiments, thepresent invention relates to compositions including one or moreisothiocyanates and/or isoselenocyanates, methods for treatment and/orprevention of pathological conditions in a subject using one or moreisothiocyanates and/or isoselenocyanates and methods for synthesis ofparticular isoselenocyanates.

A composition provided according to embodiments of the present inventionincludes one or more compounds having the structural formula:R—(CH₂)_(n)—N═C═Se, where n is an integer in the range of 1-8,inclusive, where R is an aromatic group or a non-aromatic organic group.

The term “aromatic” as used herein refers to an optionally substitutedmonocyclic or bicyclic hydrocarbon ring system containing at least oneunsaturated aromatic ring. Non-limiting examples of aromatic groupsinclude phenyl and napthyl.

In particular embodiments, compositions of the present invention arephenylalkyl isoselenocyanates having the structural formula:

where n is 1-8.

R is optionally an aromatic group substituted by one or more of thefollowing: F, Cl, Br, a lower alkyl group, a lower alkoxy group orfluorinated lower alkyl group, such as CF₃. In particular embodiments, Ris phenyl group substituted by one or more of the following: F, Cl, Br,a lower alkyl group, a lower alkoxy group or fluorinated lower alkylgroup, such as CF₃.

The term “lower alkoxy” as used herein refers to a straight chain orbranched hydrocarbon group containing from 1-4 carbon atoms which isappended to the parent molecular moiety through an oxygen atom.Illustrative examples of lower alkyl groups are methoxy, ethoxy,propoxy, 2-propoxy, butoxy and tert-butoxy.

The term “lower alkyl” as used herein refers to a straight chain orbranched hydrocarbon group containing from 1-4 carbon atoms.Illustrative examples of lower alkyl groups are methyl, ethyl,iso-propyl, n-propyl, n-butyl, iso-butyl, sec-butyl and tert-butyl.

In particular embodiments, a composition of the present inventionincludes a sulfoxide composition according to the present inventionhaving the structural formula:

where R′ is a lower alkyl group, which may be substituted orunsubstituted, branched or straight chain, and where n is an integer inthe range of 1-8, inclusive.

In certain embodiments, a composition of the present invention includesa sulfoxide composition according to the present invention having thestructural formula:

where R′ is a lower alkyl group, which may be substituted orunsubstituted, branched or straight chain, and where n is an integer inthe range of 3-8, inclusive.

In still further embodiments, a composition of the present inventionincludes a sulfoxide composition according to the present inventionhaving the structural formula:

where CH₃ can be substituted or unsubstituted and where n is an integerin the range of 3-8, inclusive.

Embodiments of a composition according to the present invention have thestructural formulas: CH₃—S(O)—(CH₂)₃—N═C═Se (termed ISC-SFN3);CH₃—S(O)—(CH₂)₄—N═C═Se (termed ISC-SFN4 and SFN Iso Se);CH₃—S(O)—(CH₂)₅—N═C═Se (termed ISC-SFN5); CH₃—S(O)—(CH₂)₆—N═C═Se (termedISC-SFN6); CH₃—S(O)—(CH₂)₇—N═C═Se (termed ISC-SFN7) andCH₃—S(O)—(CH₂)₈—N═C═Se (termed ISC-SFN8) Optionally, CH₃ is substitutedor unsubstituted.

In certain embodiments, R in the structural formula: R—(CH₂)_(n)—N═C═Se,where n is an integer in the range of 1-8, is a substituted aromaticgroup. In further embodiments, R is a phenyl group substituted at one ormore substitutable sites by Cl, Br, F, methyl, methoxy, and/or afluorinated lower alkyl group, such as CF₃.

The non-aromatic organic group optionally includes one or moreheteroatoms such as S, N, O and/or P.

In further embodiments where R is a non-aromatic organic group, R isCH₂═CH. For example, where R is CH₂═CH, an isoselenocyanate has theformula: CH₂═CH—CH₂—N═C═Se.

In a further option, the group (CH₂)_(n) in the formulaR—(CH₂)_(n)—N═C═Se, is substituted. For example, the group (CH₂)_(n) inthe formula R—(CH₂)_(n)—N═C═Se, is substituted by one or more of thefollowing: F, Cl, Br, a lower alkyl group, a lower alkoxy group orfluorinated lower alkyl group, such as CF₃.

Compositions including mixtures of two or more isoselenocyanates arealso specifically contemplated and are considered to be within the scopeof the present invention.

Structures of particular compounds described herein, along withabbreviations used, are shown below. Particular naturally occurring andsynthetic phenylalkyl isothiocyanate compounds with increasing chainlength in the left column; phenylalkyl isoselenocyanates of the presentinvention are shown in the center column; and phenylhexyl selenocyanatein the right column. Phenylhexyl selenocyanate (PHSC) is used as acontrol compound in particular tests described herein since it issimilar to ISC-6 in structure and contains selenium.

Methods for synthesis of an isoselenocyanate are provided according toembodiments of the present invention.

A process for synthesis of a compound according to embodiments of thepresent invention is shown in Scheme 1. Treatment of a phenylalkylamine(1) with ethyl formate leads to the formation of intermediate 2, whichon reaction with selenium powder in the presence of triphosgene andtriethylamine, in refluxing dichloromethane, furnishes the desiredisoselenocyanate (3).

Methods for synthesis of isothiocyanates and isoselenocyantesderivatives with a substituted phenyl ring are also provided by thepresent invention.

Substituted phenyl ring analogs of phenylalkyl isoselenocyanates aresynthesized (Scheme 2a), following substantially identical methodologiesas described in Scheme 1 for the synthesis of isoselenocyanates. In asimilar manner, substituted phenylalkyl isothiocyanates are synthesizedby treating the corresponding substituted arylalkylamine withthiophosgene and sodium hydroxide as shown in Scheme 2b. Severalcombinations of substitutions of phenyl ring (e.g. 2-, 3- or 4-chloro(Cl), bromo (Br), fluoro (—F), methyl (CH₃), methoxy (OCH₃) andtrifluoromethyl (—CF₃) substituted arylalkyl isothiocyanates andisoselenocyanates are synthesized starting from correspondingappropriately substituted 2-, 3-, and 4-Cl/Br/F/CH₃/OCH₃/CF₃-phenylalkylamines.

Methods of synthesis of isosteric selenium analogs of non-aromaticnaturally occurring isothiocyanates are provided by the presentinvention. For example, methods of synthesis of isosteric seleniumanalogs of sulforaphane i.e.,1-isoselenocyanato-4-(methylsulfinyl)butane (ISC-SFN4, also called SFNIso Se herein), are provided according to embodiments of the presentinvention. The synthetic route followed is outlined in Scheme 3. The keyintermediate 1-amino-4-(methylsulfinyl)butane (1 in scheme 3) issynthesized and subjected to a sequence of reactions as shown in Scheme3 to obtain the desired ISC-SFN4 (3). Further analogs, includingISC-SFN3, ISC-SFN6, ISC-SFN7, and ISC-SFN8 which are isosteric seleniumanalogs of corresponding naturally occurring sulfoxide isothiocyanateanalogs with varying alkyl chain length are synthesized using a similarsynthetic strategy.

In further embodiments, an allyl isoselenocyanate is synthesizedaccording to embodiments of the present invention starting fromallylamine as detailed in Scheme 4.

Conjugate Compositions

A compound of the present invention is conjugated to one or moreproperty-enhancing moieties according to embodiments of the presentinvention for modification of one or more characteristics of thecompound. The present invention provides conjugates of organicisothiocyanates and/or isoselenocyanates, in order to reduce toxicity,increase solubility and/or increase bioavailability in particularembodiments of the present invention. Methods of synthesis of suchconjugates are also provided by embodiments of the presently describedinvention.

For example, in particular embodiments, a compound of the presentinvention is conjugated to a water solubility-enhancing moiety, to yielda conjugate which is more water soluble than the compound. Thus, inparticular embodiments, water soluble isothiocyanate or isoselenocyanatecompounds of the present invention are conjugated to glutathione (GSH),cysteine (Cys) or N-acetylcysteine (NAC) to yield the correspondingGSH-, Cys-, or NAC-conjugate.

An exemplary structure of a glutathione conjugate of a phenylalkylisoselenocyanate, where n is an integer in the range of 1-8, inclusive:

An exemplary structure of a cysteine conjugate of a phenylalkylisoselenocyanate, where n is an integer in the range of 1-8, inclusive:

An exemplary structure of an N-acetylcysteine conjugate of a phenylalkylisoselenocyanate, where n is an integer in the range of 1-8, inclusive:

The NAC-conjugates of organic isoselenocyanates, such as ISC-4 andISC-6, are made by reacting corresponding isoselenocyanate withN-acetylcysteine in aqueous ethanol (50%) at room temperature undernitrogen atmosphere. The GSH or cysteine conjugates of isoselenocyanatesare synthesized following a similar procedure.

Compositions according to embodiments of the present invention preventand inhibit cancer cell multiplication and tumor development and areconsidered useful as chemotherapeutic and chemopreventive agents. Inaddition, isoselenocyanate compositions according to embodiments of thepresent invention induce cell death in cancer cells more effectivelythan corresponding isothiocyanates or derivatives thereof. Further,animal studies show significant reduction in melanoma tumor developmentby isoselenocyanates of the present invention at doses three times lowerthan those of corresponding isothiocyanates, without significanttoxicity.

Methods and compositions are provided according to the present inventionfor treating cancer. Particular cancers treated using methods andcompositions described herein are characterized by abnormal cellproliferation including, but not limited to, pre-neoplastichyperproliferation, cancer in-situ, neoplasms and metastasis. Methodsand compositions of the present invention can be used for prophylaxis aswell as amelioration of signs and/or symptoms of cancer.

A therapeutically effective amount of a composition is an amount whichhas a beneficial effect in a subject being treated. In subjects havingcancer or at risk for having cancer, such as a condition characterizedby abnormal cell proliferation including, but not limited to,pre-neoplastic hyperproliferation, cancer in-situ, neoplasms,metastasis, a tumor, a benign growth or other condition responsive to anisoselenocyanate composition, a therapeutically effective amount of acomposition is effective to ameliorate or prevent one or more signsand/or symptoms of the condition. For example, a therapeuticallyeffective amount of a composition is effective to detectably increaseapoptosis and/or decrease proliferation of cells of a cancer conditioncharacterized by abnormal cell proliferation including, but not limitedto, pre-neoplastic hyperproliferation, cancer in-situ, neoplasms,metastasis, a tumor, a benign growth or other condition responsive to anisoselenocyanate composition.

In particular embodiments, cancers treated using methods andcompositions described herein are characterized by Akt dysregulation.

Akt, a serine/threonine protein kinase also known as protein kinase B,has a stimulatory effect on cell cycle progression, cell proliferationand inhibition of apoptosis. Akt proteins, nucleic acids and signalingpathway components are described, for instance, see Testa, J. R. et al.,PNAS, 98:10983-10985; Fayard, E. et al., J. Cell Sci., 118:5675-5678,2005; Cheng, J. and S, Nicosia, (2001) AKT signal transduction pathwayin oncogenesis, in Encyclopedic Reference of Cancer, D. Schwab, Editor.2001, Springer: Berlin, Germany, p. 35-7; Datta, S. R., et al. (1999)Cellular survival: a play in three Akts. Genes Dev, 13(22): 2905-27;Fayard, E. et al. (2005) J Cell Sci, 118(Pt 24: 5675-8; Mirza, A. M.,Fayard, E. et al. (2000) 2000. 11(6: 279-92; Nicholson, K. M. and N. G.Anderson, (2002) Cell Signal, 2002, 14(5): p. 381-95; Paez, J. and W.Sellers, (2003) P13K/PTEN/Akt Pathway: A Critical Mediator of OncogenicSignaling, in Signal Transduction in Cancer, D. Frank, Editor. 2003,Kluwer Academic Publishers: Netherlands; and Testa, J. R.; P. N.Tsichlis, (2005) Oncogene, 24(50): 7391-3 and other references listedherein.

Akt family members, Akt1, Akt2 and Akt3, are activated byphosphorylation, membrane translocation, increases in gene copy numberand/or loss of a negative regulatory phosphatase, PTEN. Increasedactivation of Akt, including increased levels of Akt and/or increasedlevels of phosphorylated Akt is an indicator of Akt dysregulationassociated with proliferation and cell survival in pathogenicconditions, such as cancer.

Akt3 is active in ˜70% of melanomas. While all three Akt isoforms areexpressed in melanocytes and melanoma cells, Akt3 is the predominantlyactive family member. Dysregulated Akt3 activity in melanoma cellsreduces cellular apoptosis mediated through caspase-3, thereby promotingmelanoma tumor development.

Akt dysregulation is determined, for instance, by measurement of Aktgene copy number, Akt protein or RNA levels and/or levels ofphosphorylated Akt, in cells known or suspected to be dysplasic,pre-cancerous, cancerous, metastatic or otherwise characterized byabnormal cell proliferation compared to normal cells. Assays for Aktdysregulation include, but are not limited to immunoassays and nucleicacid assays.

Methods of treating a subject are provided according to embodiments ofthe present invention which include administering a therapeuticallyeffective amount of a composition including an isothiocyanate and/orisoselenocyanate to a subject in need thereof, wherein theisothiocyanate or isoselenocyanate has the structural formula:R—(CH₂)_(n)—N═C═X, where n is an integer in the range of 1-8, inclusive,where X is S or Se, and where R is selected from the group consistingof: an aromatic group and a non-aromatic organic group and wherein thesubject has a condition characterized by Akt dysregulation, such ascancer, pre-neoplastic hyperproliferation, cancer in-situ, neoplasms,metastasis, tumor or benign growth. In certain embodiments of methods oftreatment of a subject, contacting cells characterized by Aktdysregulation with a therapeutic amount of an isothiocyanate and/orisoselenocyanate described herein decreases a component of an Aktsignaling pathway selected from the group consisting of: an Akt1signaling pathway; an Akt2 signaling pathway; an Akt3 signaling pathway;and a combination thereof. For example, contacting the cell with anisothiocyanate and/or isoselenocyanate decreases a component of an Aktsignaling pathway selected from pAkt1, pAkt2, pAk3, pPRAS40 and acombination thereof. In embodiments of described methods, treatment of asubject with a therapeutically effective amount of the compositionincluding an isothiocyanate and/or isoselenocyanate is substantiallywithout toxic effect on cells in which Akt is not dysregulated.

Methods of treating a subject are provided according to embodiments ofthe present invention which include administering a therapeuticallyeffective amount of a composition including an isothiocyanate and/orisoselenocyanate to a subject in need thereof, wherein theisothiocyanate or isoselenocyanate has the structural formula:phenyl-(CH₂)_(n)—N═C═X, where n is an integer in the range of 1-8,inclusive, where X is S or Se, and wherein the subject has a conditioncharacterized by Akt dysregulation, such as cancer, pre-neoplastichyperproliferation, cancer in-situ, neoplasms, metastasis, tumor orbenign growth.

Methods of treating a subject are provided according to embodiments ofthe present invention which include administering a therapeuticallyeffective amount of a composition including an isothiocyanate and/orisoselenocyanate to a subject in need thereof, wherein theisothiocyanate or isoselenocyanate is BITC, PEITC, PBITC, PHITC, ISC-1,ISC-2, ISC-4 or ISG-6 and wherein the subject has a conditioncharacterized by Akt dysregulation, such as cancer, pre-neoplastichyperproliferation, cancer in-situ, neoplasms, metastasis, tumor orbenign growth.

Methods of treating a subject are provided according to embodiments ofthe present invention which include administering an effective amount ofa composition including an isoselenocyanate to a subject in needthereof.

A method of treating a subject is provided according to embodiments ofthe present invention which includes administering to a subject in needthereof a therapeutically effective amount of a an isoselenocyanatecompound having the structural formula: R—(CH₂)_(n)—N═C═Se, where n isan integer in the range of 1-8, inclusive, where R is an aromatic groupor a non-aromatic organic group.

In embodiments of the present invention, a method of treating a subjectincludes administering an effective amount of an isoselenocyanate havingthe structural formula:

where R′ is a substituted or unsubstituted, branched or straight chain,lower alkyl group, and where n is an integer in the range of 3-8,inclusive.

In embodiments of the present invention, a method of treating a subjectincludes administering an effective amount of an isoselenocyanate havingthe structural formula:

where R′ is CH₃ and where n is an integer in the range of 1-8,inclusive.

In embodiments of the present invention, a method of treating a subjectincludes administering an effective amount of an isoselenocyanate havingthe structural formula:

where R′ is CH₃ and n is 4.

In embodiments of the present invention, a method of treating a subject,includes administering an effective amount of a phenylalkylisoselenocyanate having the structural formula:

where n is 4 or 6.

Optionally, an administered isoselenocyanate is an isoselenocyanateglutathione conjugate; an isoselenocyanate cysteine conjugate; or anisoselenocyanate N-acetylcysteine conjugate.

Optionally, an administered isoselenocyanate is a pharmaceuticallyacceptable salt, ester or amide of an isoselenocyanate described herein.

Isothiocyanate and/or isoselenocyanate compositions are providedaccording to embodiments of the present invention which inhibit tumorgrowth by inhibiting an Akt signaling cascade, particularly an Akt3signaling cascade, in cells characterized by Akt dysregulation incertain embodiments.

Methods including administration of one or more isothiocyanates and/orisoselenocyanates to a subject in need thereof are provided according toparticular embodiments of the present invention which have utility, forexample, in inhibiting the Akt signaling cascade and inhibiting cancercells.

Inhibitors of the Akt signaling cascade according to embodiments of thepresent invention have utility in treatment of subject having cancer orat risk of having cancer in which Akt deregulation occurs, such as inmelanoma and other cancers including, but not limited to, cancers of theprostate, breast, brain, ovary, lung, colon, connective tissues(sarcomas) and soft tissue.

Methods of modulating an Akt protein, such as an Akt1, Akt2 and/or anAkt3 protein, in a cell are provided according to embodiments of thepresent invention which include contacting the cell with an effectiveamount of an isoselenocyanate.

Pharmaceutical compositions including an isoselenocyanate of the presentinvention are also provided according to embodiments of the presentinvention.

A pharmaceutical composition includes an isoselenocyanate of the presentinvention and a pharmaceutically acceptable carrier in particularembodiments of the present invention. The term “pharmaceuticallyacceptable carrier” refers to a carrier which is substantially non-toxicto a subject to which the composition is administered and which issubstantially chemically inert with respect to a selenium-containingcompound of the present invention.

A pharmaceutical composition according to the invention generallyincludes about 0.1-99% of an isoselenocyanate of the present invention.Combinations of isoselenocyanates in a pharmaceutical composition arealso considered within the scope of the present invention.

Optionally, a method of treating a subject having cancer or at risk ofhaving cancer further includes an adjunct anti-cancer treatment. Anadjunct anti-cancer treatment can be administration of an anti-canceragent.

Anti-cancer agents are described, for example, in Goodman et al.,Goodman and Gilman's The Pharmacological Basis of Therapeutics, 8th Ed.,Macmillan Publishing Co., 1990.

Anti-cancer agents illustratively include acivicin, aclarubicin,acodazole, acronine, adozelesin, aldesleukin, alitretinoin, allopurinol,altretamine, ambomycin, ametantrone, amifostine, aminoglutethimide,amsacrine, anastrozole, anthramycin, arsenic trioxide, asparaginase,asperlin, azacitidine, azetepa, azotomycin, batimastat, benzodepa,bicalutamide, bisantrene, bisnafide dimesylate, bizelesin, bleomycin,brequinar, bropirimine, busulfan, cactinomycin, calusterone,capecitabine, caracemide, carbetimer, carboplatin, carmustine,carubicin, carzelesin, cedefingol, celecoxib, chlorambucil, cirolemycin,cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine,dacarbazine, dactinomycin, daunorubicin, decitabine, dexormaplatin,dezaguanine, dezaguanine mesylate, diaziquone, docetaxel, doxorubicin,droloxifene, dromostanolone, duazomycin, edatrexate, eflomithine,elsamitrucin, enloplatin, enpromate, epipropidine, epirubicin,erbulozole, esorubicin, estramustine, etanidazole, etoposide, etoprine,fadrozole, fazarabine, fenretinide, floxuridine, fludarabine,fluorouracil, fluorocitabine, fosquidone, fostriecin, fulvestrant,gemcitabine, hydroxyurea, idarubicin, ifosfamide, ilmofosine,interleukin II (IL-2, including recombinant interleukin II or rIL2),interferon alfa-2a, interferon alfa-2b, interferon alfa-nl, interferonalfa-n3, interferon beta-Ia, interferon gamma-Ib, iproplatin,irinotecan, lanreotide, letrozole, leuprolide, liarozole, lometrexol,lomustine, losoxantrone, masoprocol, maytansine, mechlorethaminehydrochloride, megestrol, melengestrol acetate, melphalan, menogaril,mercaptopurine, methotrexate, metoprine, meturedepa, mitindomide,mitocarcin, mitocromin, mitogillin, mitomalcin, mitomycin, mitosper,mitotane, mitoxantrone, mycophenolic acid, nelarabine, nocodazole,nogalamycin, ormnaplatin, oxisuran, paclitaxel, pegaspargase,peliomycin, pentamustine, peplomycin, perfosfamide, pipobroman,piposulfan, piroxantrone hydrochloride, plicamycin, plomestane,porfimer, porfiromycin, prednimustine, procarbazine, puromycin,pyrazofurin, riboprine, rogletimide, safingol, semustine, simtrazene,sparfosate, sparsomycin, spirogermanium, spiromustine, spiroplatin,streptonigrin, streptozocin, sulofenur, talisomycin, tamoxifen,tecogalan, tegafur, teloxantrone, temoporfin, teniposide, teroxirone,testolactone, thiamiprine, thioguanine, thiotepa, tiazofurin,tirapazamine, topotecan, toremifene, trestolone, triciribine,trimetrexate, triptorelin, tubulozole, uracil mustard, uredepa,vapreotide, verteporfin, vinblastine, vincristine sulfate, vindesine,vinepidine, vinglycinate, vinleurosine, vinorelbine, vinrosidine,vinzolidine, vorozole, zeniplatin, zinostatin, zoledronate, andzorubicin.

An adjunct anti-cancer treatment can be a radiation treatment of asubject or an affected area of a subject's body.

Pharmaceutical compositions suitable for delivery to a subject may beprepared in various forms illustratively including physiologicallyacceptable sterile aqueous or nonaqueous solutions, dispersions,suspensions or emulsions, and sterile powders for reconstitution intosterile injectable solutions or dispersions. Examples of suitableaqueous and nonaqueous carriers include water, ethanol, polyols such aspropylene glycol, polyethylene glycol, glycerol, and the like, suitablemixtures thereof; vegetable oils such as olive oil; and injectableorganic esters such as ethyloleate. Proper fluidity can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants, such as sodium lauryl sulfate. Additionalcomponents illustratively including a buffer, a solvent, or a diluentmay be included.

Such formulations are administered by a suitable route includingparenteral and oral administration. Administration may include systemicor local injection, and particularly intravenous injection.

These compositions may also contain adjuvants such as preserving,wetting, emulsifying, and dispensing agents. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. It may also be desirable to include isotonic agents, forexample, sugars, sodium chloride, and substances similar in nature.Prolonged delivery of an injectable pharmaceutical form can be broughtabout by the use of agents delaying absorption, for example, aluminummonostearate and gelatin.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms,selenium-containing compound of the present invention is admixed with atleast one inert customary excipient (or carrier) such as sodium citrateor dicalcium phosphate or (a) fillers or extenders, as for example,starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b)binders, as for example, carboxymethylcellulose, alignates, gelatin,polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, as forexample, glycerol, (d) disintegrating agents, as for example, agar-agar,calcium carbonate, plant starches such as potato or tapioca starch,alginic acid, certain complex silicates, and sodium carbonate, (e)solution retarders, as for example, paraffin, (f) absorptionaccelerators, as for example, quaternary ammonium compounds, (g) wettingagents, as for example, cetyl alcohol, glycerol monostearate, andglycols (h) adsorbents, as for example, kaolin and bentonite, and (i)lubricants, as for example, talc, calcium stearate, magnesium stearate,solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof.In the case of capsules, tablets, and pills, the dosage forms may alsoinclude a buffering agent.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethyleneglycols, andthe like.

Solid dosage forms such as tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells, such as entericcoatings and others well known in the art. They may contain opacifyingagents, and can also be of such composition that they release the activecompound or compounds in a certain part of the intestinal tract in adelayed manner. Examples of embedding compositions which can be used arepolymeric substances and waxes. The active compounds can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-mentioned excipients.

Liquid dosage forms for oral administration include a pharmaceuticallyacceptable carrier formulated as an emulsion, solution, suspension,syrup, or elixir. In addition to the active compounds, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, as for example,ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol,dimethylformamide, oils, in particular, cottonseed oil, groundnut oil,corn germ oil, olive oil, castor oil and sesame oil, glycerol,tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters ofsorbitan or mixtures of these substances, and the like.

Besides such inert diluents, the composition can also include adjuvants,such as wetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents.

Suspensions, in addition to an inventive conjugate, may containsuspending agents, as for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitol esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar or tragacanth,or mixtures of these substances, and the like.

In particular embodiments, compositions of the present invention areformulated for topical application. In further particular embodiments,compositions of the present invention are formulated for topicalapplication and are characterized by less than 10% absorption of anactive ingredient in the composition into the system of an individualtreated topically. In still further particular embodiments, compositionsof the present invention are formulated for topical application and arecharacterized by less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% absorbtionof an active ingredient in the composition into the system of anindividual treated topically. Absorption into the system of anindividual can be measured by any of various methods, particularly assayfor the active ingredient, a metabolite and/or a breakdown product ofthe active ingredient in a sample obtained from an individual treatedwith the topical formulation. For example, a blood, plasma or serumsample can be assayed for presence of the active ingredient, ametabolite of the active ingredient and/or a breakdown product of theactive ingredient.

A topical formulation can be an ointment, lotion, cream or gel inparticular embodiments. Topical dosage forms such as ointment, lotion,cream or gel bases are described in Remington: The Science and Practiceof Pharmacy, 21^(st) Ed., Lippincott Williams & Wilkins, 2006, p.880-882 and p. 886-888; and in Allen, L. V. et al., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, 8^(th) Ed.,Lippincott Williams & Wilkins, 2005, p. 277-297.

Pharmaceutically acceptable carriers and formulation of pharmaceuticalcompositions are known in the art, illustratively including, but notlimited to, as described in Remington: The Science and Practice ofPharmacy, 21^(st) Ed., Lippincott, Williams & Wilkins, Philadelphia,Pa., 2006; and Allen, L. V. et al., Ansel's Pharmaceutical Dosage Formsand Drug Delivery Systems, 8^(th) Ed., Lippincott, Williams & Wilkins,Philadelphia, Pa., 2005.

The term subject refers to an individual in need of treatment for apathological condition, particularly cancer, and generally includesmammals and birds, such as, but not limited to, humans, other primates,cats, dogs, cows, horses, rodents, pigs, sheep, goats and poultry.

A pharmaceutical composition according to the present invention issuitable for administration to a subject by a variety of systemic and/orlocal routes including, but not limited to, intravenous, intramuscular,subcutaneous, intraperitoneal, oral, otic, rectal, vaginal, topical,parenteral, pulmonary, ocular, nasal, intratumoral and mucosal.

An inventive composition may be administered acutely or chronically. Forexample, a composition as described herein may be administered as aunitary dose or in multiple doses over a relatively limited period oftime, such as seconds-hours. In a further embodiment, administration mayinclude multiple doses administered over a period of days-years, such asfor chronic treatment of cancer.

With regard to administration of isoselenocyanates to a mammaliansubject, particular exemplary effective dosage ranges withoutsignificant systemic toxicity are described in terms of amounts ofselenium administered via administration of the isoselenocyanate. Thus,for example, when delivered by a parenteral route, such asintraperitoneal or intravenous, an exemplary therapeutically effectivedosage of an isoselenocyanate is in the range of about 1-4 ppm selenium,administered three times per week. It is noted that the dose range“about 1-4 ppm selenium” refers to a dose of “about 1 mg/kg-4 mg/kg ofselenium.” For example, a dose of 3 ppm selenium when referring to ISC-4is equivalent to a dose of 9.1 mg/kg of ISC-4. Similarly, a dose of 3ppm selenium when referring to ISC-6 is equivalent to a dose of 10.17mg/kg of ISC-6. In a further example, when delivered topically, anexemplary therapeutically effective dosage of ISC-4 or ISC-6 is in therange of about 0.1-1 ppm selenium, administered daily. In a furtherexample, when delivered orally, an exemplary therapeutically effectivedosage of an isoselenocyanate is in the range of about 1-15 ppmselenium.

A therapeutically effective amount of a pharmaceutical compositionaccording to the present invention will vary depending on the particularpharmaceutical composition used, the severity of the condition to betreated, the species of the subject, the age and sex of the subject andthe general physical characteristics of the subject to be treated. Oneof skill in the art could determine a therapeutically effective amountin view of these and other considerations typical in medical practice.In general it is contemplated that a therapeutically effective amountwould be in the range of about 0.001 mg/kg-100 mg/kg body weight,optionally in the range of about 0.01-10 mg/kg, and further optionallyin the range of about 0.1-5 mg/kg. Further, dosage may be adjusteddepending on whether treatment is to be acute or continuing.

Advantageously, anti-cancer compounds according to embodiments of thepresent invention are formulated to achieve lipid-solubility and/oraqueous-solubility.

In particular embodiments, a pharmaceutically acceptable carrier is aparticulate carrier such as lipid particles including liposomes,micelles, unilamellar or mulitlamellar vesicles; polymer particles suchas hydrogel particles, polyglycolic acid particles or polylactic acidparticles; inorganic particles such as calcium phosphate particles suchas described in for example U.S. Pat. No. 5,648,097; andinorganic/organic particulate carriers such as described for example inU.S. Pat. No. 6,630,486.

A particulate pharmaceutically acceptable carrier can be selected fromamong a lipid particle; a polymer particle; an inorganic particle; andan inorganic/organic particle. A mixture of particle types can also beincluded as a particulate pharmaceutically acceptable carrier.

A particulate carrier is typically formulated such that particles havean average particle size in the range of about 1 nm-10 microns. Inparticular embodiments, a particulate carrier is formulated such thatparticles have an average particle size in the range of about 1 nm-100nm.

Embodiments of inventive compositions and methods are illustrated in thefollowing examples. These examples are provided for illustrativepurposes and are not considered limitations on the scope of inventivecompositions and methods.

EXAMPLES Example 1 Cell Lines and Culture Conditions

The human metastatic melanoma cell lines UACC 903 and 1205 Lu; normalhuman fibroblast cells (FF2441) are maintained in DMEM (Invitrogen,Carlsbad, Calif.) supplemented with 10% FBS (Hyclone, Logan, Utah).Vertical growth phase (VGP) melanoma cell line WM115 is maintained inTu2% medium lacking calcium chloride, supplemented with 2% heat treated(56° C. for 30 minutes) FBS and L-glutamine (Mediatech, Handon, Va.) asdescribed in Stahl J M, et al., Cancer Res 2004; 64:7002-10.

Colon adenocarcinoma cell line (Caco-2, ATCC No. HTB-37) is grown eitherin Advanced DMEM supplemented with 10% heat treated (56° C. for 30minutes) FBS and L-glutamine. Fibrosarcoma (HT-1080; ATCC No. CCL-121),prostate adenocarcinoma (PC-3; ATCC No. CRL-1435), breast adenocarcinomacell line (MDA-MB-231; ATCC No. HTB-26), glioblastoma cell line (T98G;ATCC No. CRL-1690) and human melanoma cell line UACC903 are grown inDMEM supplemented with 10% FBS.

Example 2 Chemical Synthesis

Synthetic methods described in Examples 2-12 refer generally to thefollowing numbered structures:

Melting points were recorded on a Fisher-Johns melting point apparatusand are uncorrected. Unless stated otherwise, proton NMR spectra wererecorded in a Bruker AM 360WB instrument using CDCl₃ as solvent. Thechemical shifts are reported in ppm downfield from TMS. High-resolutionMS (EI) are determined at the Chemistry Instrumentation Center, StateUniversity of New York at Buffalo, N.Y. Thin-layer chromatography (TLC)is developed on aluminum-supported, pre-coated silica gel plates (EMIndustries, Gibbstown, N.J.). Column chromatography was conducted onsilica gel (60-200 mesh). Benzyl isothiocyanate (RITC, 1a), phenylethylisothiocyanate (PEITC, 1b), and phenylbutylisothiocyanate (PBITC, 1c)are obtained from commercial sources. Phenylhexylisothiocyanate (PHITC,1d) is synthesized as described in Morse, M. A. et al., Cancer Res 1991,51, (7), 1846-50.

Example 3

A general method for the synthesis of phenylalkylformamides is describedin Elliott, M. C.; Williams, E., Synthesis and reactions of partiallyreduced biisoquinolines. Org Biomol Chem 2003, 1, (17), 3038-47. Ethylformate (120 mmol) was added dropwise to phenylalkylamine (40 mmol) atroom temperature and the resulting mixture was refluxed for 4-6 h. Theexcess ethyl formate was removed under reduced pressure to yield thecorresponding phenylakylformamide as an oil.

Phenylethylformamide: (AS3.090) ¹H NMR (CDCl₃) δ 2.84 (t, 2H, J=6.9 Hz),3.57 (dt, 2H, J=6.9 Hz and 6.6 Hz), 5.68 (br d, 1H, NH), 7.15-7.35 (m,5H), 8.12 (s, 1H, CHO); HRMS (EI) calcd for C₉H₁₁NO, 149.0835; found,149.0839.

Phenylbutylformamide. ¹H NMR (CDCl₃) δ 1.56-1.62 (m, 2H), 1.66-1.72 (m,2H), 2.66 (t, 2H, J=6.5 Hz), 3.35 (dt, 2H, J=7.0 and 6.5 Hz), 5.92 (brs, 1H), 7.18-7.24 (m, 2H), 7.29-7.33 (m, 2H), 8.19 (s, 1H); HRMS (EI)calcd for C₁₁H₁₅NO, 177.1148; found, 177.1149.

Phenylhexylformamide. ¹H NMR (CDCl₃) δ 1.36-1.41 (m, 4H), 1.52-1.58 (m,2H), 1.61-1.67 (m, 2H), 2.63 (t, 2H, J=7.5 Hz), 3.30 (dt, 2H, J=7.0 and6.5 Hz), 5.58 (br s, 1H), 7.18-7.21 (m, 3H), 7.28-7.31 (m, 2H), 8.19 (s,1H); HRMS (EI) calcd for C₁₃H₁₉NO, 205.1461; found, 205.1462.

Example 4

Isoselenocyanates are synthesized using a modified method described inFernández-Bolaños, J. G., López, O., Ulgar, V., Maya, I., and Fuentes,J., Synthesis of O-unprotected glycosyl selenoureas. A new access tobicyclic sugar isoureas. Tetrahedron Lett. 2004, 45, 4081-4084. Solidtriphosgene is used in a one-pot dehydration of the formamides inrefluxing dichloromethane (Scheme 5).

Scheme 5 shows synthesis of compounds 1 and 2; Reagents and conditions:(a) C₂H₅OCHO, −20° C. to reflux (b) Et₃N, triphosgene, Se powder,CH₂Cl₂, reflux (c) CSCl₂, NaOH.

General Experimental Procedure for the Synthesis of ISC Compounds

The synthetic strategy involves the formylation of phenylalkylamines,followed by treatment with triphosgene and selenium powder in thepresence of triethylamine to furnish the desired phenylalkylisoselenocyanates (2) in good yields as oils.

The compounds are purified by silica gel column chromatography and arecharacterized on the basis of NMR and high-resolution MS data. (Pureisolated compounds are light yellow (ISC-1 and ISC-2) to colorless(ISC-4 and ISC-6) which tend to get a little darker after storing forlonger time).

In a particular example, to a refluxing mixture of the aryl alkylformamides (1.5 mmol), triethylamine (6.4 mmol) in CH₂Cl₂ (5 mL) and 4 Åmolecular sieves was added dropwise a solution of triphosgene (0.8 mmol)in CH₂Cl₂ (2 mL) for a period of 1 hour. After the addition wascomplete, the mixture was refluxed for an additional 2.5 hour. Seleniumpowder (3.0 mmol) was then added and the resulting mixture was refluxedfor other 6-8 hours. The mixture is cooled, filtered, and the solventwas evaporated to yield the crude mixture, which is purified by silicagel column chromatography to afford isoselenocyanates.

In a further example, a solution of triphosgene (5.0 mmol) in CH₂Cl₂ (15mL) is added over a 1 hour period to a refluxing mixture ofphenylalkylformamides (10.0 mmol), triethylamine (43.0 mmol) and 4 Åmolecular sieves in CH₂Cl₂ (35 mL). The mixture is then refluxed for anadditional 2.5 hours. Selenium powder (20 mmol) is then added andresulting mixture refluxed for 6-8 hours. Mixture is cooled, filtered,and solvent evaporated yielding a crude mixture, which is purified bysilica gel column chromatography generating pure isoselenocyanates.Isothiocyanates and isoselenocyanates are >99% pure.

Phenylalkyl isothiocyanates are obtained commercially (BITC, PEITC, andPBITC). PHITC is synthesized as described in Morse, M. A. et al., CancerRes 1991, 51, (7), 1846-50. Phenylhexylamine (3d) required for thesynthesis of 1d is synthesized by converting phenylhexyl chloride to thecorresponding azide by treatment with sodium azide in DMF, followed bygeneration of the 3d by reduction of azide with lithium aluminiumhydride as described in Gopalakrishnan, G. et al., J. Labelled. Comp.Radiopharma. 1988, 25, (4), 383-393.

Example 5 Benzyl Isoselenocyanate (2a)

To a refluxing mixture of the formamides (1.5 mmol), triethylamine (6.4mmol) in CH₂Cl₂ (5 mL) and 4 Å molecular sieves was added dropwise asolution of triphosgene (0.8 mmol) in CH₂Cl₂ (2 mL) for a period of 1 h.After the addition was complete, the mixture was refluxed for anadditional 2.5 h. Selenium powder (3.0 mmol) was then added and theresulting mixture was refluxed for other 6 h. The mixture was cooled,filtered, and the solvent was evaporated to yield the crude mixture,which was purified by silica gel column chromatography to affordisoselenocyanates. (AS3.094): viscous oil, ¹H NMR (CDCl₃) δ 4.81 (s, 2H,CH₂), 7.30-7.44 (m, 5H); HRMS (EI) calcd for C₈H₁₇NSe, 196.9738; found,196.9741.

Example 6 Phenylethyl Isoselenocyanate (2b)

(A53.091) A mixture of phenylethylformamide (g, 1.5 mmol), triethylamine(6.4 mmol), 4 Å molecular sieves (g), triphosgene (0.8 mmol), andselenium powder (3.0 mmol) in CH₂Cl₂ was refluxed and worked up asmentioned for 2a. The crude residue thus obtained was purified by silicagel column chromatography (EtOAc/hexanes 2:98) to give 0.96 g (95%) of2b as an oil. ¹NMR (CDCl₃) δ 3.03 (t, 2H, J=6.9 Hz), 3.81 (t, 2H, J=6.9Hz) 7.20-7.38 (m, 5H); HRMS (EI) calcd for C₉H₉NSe, 210.9895; found,210.9892.

Example 7 Phenylbutyl Isoselenocyanate (2c)

A mixture of phenylbutyl formamide (g, 1.5 mmol), triethylamine (6.4mmol), 4 Å molecular sieves (g), triphosgene (0.8 mmol), and seleniumpowder (3.0 mmol) in CH₂Cl₂ was refluxed and worked up as mentioned for2a. The crude residue thus obtained was purified by silica gel columnchromatography (EtOAc/hexanes 2:98) to give 0.96 g (95%) of 2c as anoil. ¹H NMR (CDCl₃) δ 1.74-1.76 (m, 4H), 2.66 (t, 2H, J=6.6 Hz), 3.60(t, 21-1, J=5.6 Hz), 7.15-7.32 (m, 5H); HRMS (EI) calcd for C₁₁H₁₃NSe,239.0208; found, 239.0211.

Example 8 Phenylhexyl Isoselenocyanate (2d)

A mixture of phenylbutyl formamide (g, 1.5 mmol), triethylamine (6.4mmol), 4 Å molecular sieves (g), triphosgene (0.8 mmol), and seleniumpowder (3.0 mmol) in CH₂Cl₂ was refluxed and worked up as mentioned for2a. The crude residue thus obtained was purified by silica gel columnchromatography (EtOAc/hexanes 2:98) to give 0.96 g (95%) of 2d as anoil. ¹H NMR (CDCl₃) δ 1.37-1.43 (m, 2H), 1.45-1.51 (m, 2H), 1.67 (dt,2H, J=15.2 and 7.6 Hz), 1.75 (dt, 2H, J=14.9 and 6.7 Hz), 2.64 (t, 2H,J=7.6 Hz), 3.60 (t, 2H, J=6.7 Hz), 7.19-7.25 (m, 3H), 7.32-7.40 (m, 2H);HRMS (EI) calcd for C₁₃H₁₇NSe, 267.0521; found, 267.0529.

Example 9 ISC-4-NAC Conjugate (5)

Scheme 6 shows the synthesis of NAC conjugate (5) of ISC-4. The IC₅₀ of5 (UACC 903M cells)=17±2.

Example 10 1-isoselenocyanato-4-(methylsulfinyl)butane (ISC-SFN4, AlsoCalled SFN Iso Se Herein)

viscous oil; ¹H NMR in CDCl₃: 3.72 (t, 2H, J=6.53 Hz, N—CH₂), 3.10 (t,2H, J=6.94 Hz, SO—CH₂), 2.97 (s, 3H, S—CH₃), 1.98-2.07 (m, 4H,C—CH₂—CH₂—C).

Example 11 IC₅₀ Values of ITC and ISC Compounds

To measure the inhibitory potency (IC₅₀) of isothiocyanate andisoselenocyanate derivatives in various cancer cells, MTS (CellTiter 96Aqueous Non Radioactive Cell Proliferation Assay kit, Promega, Madison,Wis.) is used. Cellular viability is quantified by MTS assay and doseresponse curves plotted. 5×10³ melanoma cells (UACC 903, 1205 LU orWM115) cells per well in 100 μL DMEM containing 10% FBS are grown in a96-well plate for 24 h and then treated with increasing concentrationsof the indicated isothiocyanate or isoselenocyanate for 24 h. After 24hours of treatment viable cells are measured by reading soluble coloredformazan product at 490 nm using a microplate reader. Results of 3independent experiments are considered for the determination of IC₅₀.The IC₅₀ values are calculated from the concentration curves using GraphPad Prism software and are shown in Table I for cells and compoundsindicated.

TABLE I IC₅₀ (μm) 24 h drug treatment Cell ISC- ISC- ISC- ISC- Line BITCPEITC PBITC PHITC 1 2 4 6 UACC 15 ± 12 ± 1 15 ± 1 15 ± 2 16 ± 12 ± 12 ±10 ±  903 3 3 4 3 1 1205 >25 24 ± 2 19 ± 5 11 ± 4 >25 18 ± 11 ± 7 ± Lu 41 4 WM 13 ± 12 ± 1  7 ± 1  8 ± 1 >25  8 ±  7 ± 7 ± 115 1 1 2 2

Table I shows a comparison of the IC₅₀ of isothiocyanates andisoselenocyanates in three independently derived melanoma cell lines,UACC 903, 1205 Lu and WM115. A general trend is observed in whichincreasing carbon chain length and substitution of selenium for sulfurdecreased the IC₅₀. Increased potency ranged from 30-70% with increasingchain length and/or sulfur substituted for selenium. Thus,isothiocyanate analogs with longer alkyl chain lengths and sulfursubstituted for selenium are better inhibitors of cultured melanomacells.

Example 12 SAR Study on Cancer Cell Lines

Certain ITCs (1) and the corresponding ISCs (2) are tested for theirability to inhibit cell growth in five cancer cell lines e.g. melanoma,breast, glioblastoma, fibrosarcoma, colon and prostate cancers. In vitroinhibitory efficacy of cancer cell lines representing different cancertypes following treatment with ITC and ISC is measured using the3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) assay (Promega, Madison, Wis.). In brief, 2.5-5×10³ cells per wellin 100 μL DMEM containing 10% FBS are grown in a 96-well plate for 24 hand treated with either control DMSO vehicle or increasingconcentrations (2.5-100 μM) of ITC and ISC for 24 hours. At this pointcells are treated individually with either vehicle control DMSO or withincreasing concentrations of ITC or ISC (2.5-100 μM) for 24 h. Thepercentages of viable cells compared to control DMSO treated cells aredetermined using MTS assay and IC₅₀ values calculated using GraphPadPrism version 4.01 (GraphPad software, San Diego, Calif.). IC₅₀ valuefor each compound was determined by at least three independentexperiments and represented with a standard error (Table II).

The IC₅₀ values for compounds 1 and 2 are depicted in Table II. Thevalues consistently decreased with increasing alkyl chain length of ITCsin case of MDA-MB-231, T98G, and PC-3 cell lines; but showed noparticular trend in case of HT-1080, Caco-2, and UACC 903 cells. ISC-1was least effective in killing cells in all the cell lines testedcompared to its higher alkyl chain analogs ISC-2 to ISC-6. Among ISC-2,ISC-4 and ISC-6 there was no particular trend. The values generallydecreased with increasing chain length for all the cancer cell linestested except breast cancer cells MDA-MB 231 in case of ISC compoundsand sarcoma HT 1080 cells in case of ITCs. Except for ISC-1 and ISC-2 inUACC 903 cells, the ISC derivatives had lower IC₅₀ values thancorresponding ITCs.

TABLE II IC₅₀ (μM) of ITC and ISC derivatives on different cancer cellsCancer cell lines IC₅₀ (μM) Breast Glioblastoma Prostate FibrosarcomaColon Melanoma Compounds MDA-MB-231 T-98-G PC-3 HT-1080 Caco-2 UACC 9031a (BITC) 42 ± 3 >100 >50 >50 15 ± 2 15 ± 3 1b (PEITC) 38 ± 6 >100 24 ±2 15 ± 1 14 ± 2 12 ± 1 1c (PBITC) 27 ± 2 35 ± 1 24 ± 2 15 ± 1 27 ± 2 15± 1 1d (PHITC) 24 ± 2 26 ± 2 17 ± 1 29 ± 3 49 ± 9 15 ± 1 2a (ISC-1) 29 ±2 43 ± 4 24 ± 1 13 ± 3 13 ± 3 16 ± 3 2b (ISC-2) 20 ± 3 24 ± 1 16 ± 1 12± 3 11 ± 1 12 ± 4 2c (ISC-4)  21 ± 64 27 ± 1 19 ± 1 11 ± 1 12 ± 3 12 ± 32d (ISC-6) 22 ± 2 23 ± 2 14 ± 1 12 ± 1 10 ± 1 10 ± 1 Values are mean ±S.E. * Drug treatment for 72 h

LogP values are estimated for isothiocyanates and isoselenocyanatesusing ChemDraw 9.0 Ultra and these values are compared in Table III.

TABLE III ITCs CLogP ISCs CLogP 1a (BITC) 3.204 2a (ISC-1) 3.177 1b(PEITC) 3.263 2b (ISC-2) 3.506 1c (PBITC) 4.171 2c (ISC-4) 4.414 1d(PHITC) 5.229 2d (ISC-6) 5.472 ^(a)LogP was estimated using ChemDraw 9.0Ultra

Example 13

Cell viability of melanoma cells following treatment with isothiocyanateor an isoselenocyanate is measured using the3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) assay (Promega, Madison, Wis.).

Briefly, 5×10³ melanoma cells (UACC 903, 1205 LU or WM115) or humanfibroblast (FF2441) cells per well in 100 μL DMEM containing 10% FBS aregrown in a 96-well plate for 24 h and treated with either control DMSOvehicle; phenylhexyl selenocyanate (PHSC), used as a control compoundsince it is similar to ISC-6 in structure and contains selenium; API-2,an Akt inhibitor used for comparison purposes; or increasingconcentrations (2.5-50 μM) of an isothiocyanate or an isoselenocyanatefor 24 h. Cellular viability compared to control treated cells ismeasured using the MTS assay. IC₅₀ values for each compound inrespective cell lines is determined from three independent experimentsusing GraphPad Prism version 4.01 (GraphPad software, San Diego,Calif.).

FIG. 1A shows a comparison of cell viability following exposure to PBITCor ISC-4, compared to controls. FIG. 1B shows a comparison of cellviability following exposure to PHITC or ISC-6, compared to controls.Cell viability is determined using MTS assay. ISC-4 effectively reducescell viability at concentrations of 10 μM and 15 μM (IC₅₀=12±3 and 10±1μM) compared to controls (API-2, PHSC) or PBITC or PHITC (IC₅₀=15±1 and15±2 μM). The average value represented as the percentage of controlDMSO treated cells. Phenylhexyl selenocyanate (PHSC) is used as acontrol compound since it is similar to ISC-6 in structure and containsselenium. API-2 is an Akt inhibitor used for comparison purposes. Thus,ISC-4 and ISC-6 are more effective at inhibiting growth of melanomacells than sulfur containing PBITC, PHITC, control PHSC or API-2.

Example 14

Cellular proliferation in vitro is measured by seeding 5×10³ cells in96-well plate, followed by treatment for 24 hours with vehicle controlDMSO, or 5 μM, 10 μM or 15 μM of API-2 (a known Akt inhibitor), PHSC,PBITC, PHITC, ISC-4, or ISC-6. Proliferation rate is measured using aBrdU ELISA kit (Roche Applied Sciences, Indianapolis, Ind.).

FIG. 2A shows results of proliferation analysis of UACC 903 cellstreated with DMSO or different concentrations (5 uM, 10 uM, 15 uM) ofAPI-2, PHSC, PBITC or ISC-4 for 24 hours. FIG. 2B shows results ofproliferation analysis of UACC 903 cells treated with DMSO or differentconcentrations (5 uM, 10 uM, 15 uM) of API-2, PHSC, PHITC or ISC-6 for24 hours. BrdUrd is used to label cells for 4-6 hours. At 15 μMconcentration, ISC-4 and ISC-6 reduced UACC 903 cellular proliferativepotential by ˜80-90% compared to controls. Results represent the averageof 3 independent experiments; bars represent SEM. The average value isrepresented as the percentage of control DMSO treated cells.

In contrast to the Akt inhibitor API-2 or the control 6-carbon seleniumcompound PHSC, increasing concentrations of PBITC, ISC-4, PHITC or ISC-6led to decreased proliferative potential of treated cells. Both ISC-4and ISC-6 are ˜2-fold more effective than PBITC or PHITC at inhibitingcellular proliferation.

Example 15

Apoptosis rates are measured by seeding 5×10³ cells in 96-well plate,followed by treatment for 24 hours with vehicle control DMSO; API-2;PHSC; PBITC; PHITC; ISC-4; or ISC-6. Apoptosis rates are measured usingan Apo-ONE Homogenous caspase-3/7 Assay kit (Promega Corporation,Madison, Wis.).

FIG. 3A shows the effects of treatment of UACC 903 cells in culture withDMSO or 5 μM, 10 μM or 15 μM of API-2, PHSC, PBITC or ISC-4 for 24 hourson caspase-3/7 activity, an indicator of apoptosis. FIG. 3B shows theeffects of treatment of UACC 903 cells in culture with DMSO or 5 μM, 10μM or 15 μM of API-2, PHSC, PHITC or ISC-6 for 24 hours on caspase-3/7activity, an indicator of apoptosis. The isoselenocyanate compoundspromote apoptosis in melanoma cells. Levels of caspase-3/7 activity incells exposed to API-2, PHSC, PBITC, PHITC, ISC-4 or ISC-6 are measuredusing the Apo-ONE homogeneous caspase-3/7 assay kit. The graphs in FIGS.3A and 3B show fold increase in caspase-3/7 activity relative to DMSOvehicle treated cells on the x-axis. Results represent average of 3independent experiments. Bars indicate SEM. In contrast to API-2 andPHSC, increasing concentrations of PBITC, ISC-4, PHITC and ISC-6increase cellular apoptosis of UACC 903 melanoma cells as shown in FIGS.3A and 3B. ISC-4 and ISC-6 are ˜2-fold more effective than PBITC atinducing apoptosis.

Example 16

Cell cycle analysis is performed by plating 1.5×10⁶ melanoma cells in100-mm culture dish. Two days following plating, cells are treated withDMSO, or 5 μM, 10 μM or 15 μM of API-2, PHSC, PBITC, PHITC, ISC-4, orISC-6 for 24 hours. Cells are collected by trypsinization and stainedusing propidium iodide (13). Trypsinized cells are centrifuged (500×g,for 5 minutes) and treated with 1 mL of propidium iodide stainingsolution (100 μg/mL; Sigma, St Louis, Mo.), 20 μg/mL Ribonuclease A(Roche Applied Sciences, Indianapolis, Ind.) 3 μg/mL Triton X-100dissolved in 0.1% (W/V) sodium citrate for 30 minutes at 4° C. Stainedcells are analyzed using the FACScan analyzer (Becton Dickinson, SanJose, Calif.) and data analyzed using ModFit LT software (VeritySoftware House, Topsham, Me.).

FIG. 4A shows results of cell cycle analysis of UACC 903 cells treatedwith controls (API-2, PHSC), PBITC or ISC-4, or, DMSO vehicle control.FIG. 4B shows results of cell cycle analysis of UACC 903 cells treatedwith controls (API-2, PHSC), PHITC or ISC-6, or, DMSO vehicle control.ISC-4, ISC-6, PBITC or PHITC treatment increased the sub G0/G1 cellpopulation (an indicator of apoptosis) and induces G2/M cell cyclearrest in melanoma cells compared to DMSO or controls API-2 and PHSC.Results represent average of 2 independent experiments. Enhancedapoptosis induced by ISC-4 or ISC-6 is confirmed through cell cycleanalysis of asynchronously growing UACC 903 cells. A significantincrease is observed in the sub G0/G1 population in PBITC, ISC-4, PHITCor ISC-6 treated cells which is indicative of cells undergoingapoptosis.

Table IV shows results of cell cycle analysis following treatment withcontrols compounds DMSO, API-2 or PHSC or test compounds PBITC, PHITC,ISC-4 or ISC-6. Treated cells are stained with propidium iodide and cellcycle analyzed using a FACScan analyzer such that the proportion ofcells in each phase of cell cycle (G0/G1, S, G2/M) is estimated. TheG2/M cell population increases 2-3 fold following 15 μM PBITC, PHITC,ISC-4 or ISC-6 exposure. A significant decrease in G0/G1 cell populationoccurred following ITC or ISC treatment. Enhanced apoptosis induced byISC-4 or ISC-6 is confirmed through cell cycle analysis ofasynchronously growing UACC 903 cells. Analysis of cells in each stageof the cell cycle showed decreasing numbers of cells in the S— and G1/G0phase with a corresponding increase in the G2/M phase.

TABLE IV UACC 903—Cell Cycle Analysis Following Treatment With ISC-4 orISC-6 and Corresponding Isothiocyanates Concen- tration API-2 PHSC PBITCISC-4 PHITC ISC-6 (μM) DMSO 15 15 5 10 15 5 10 15 5 10 15 5 10 15 subG0/0.4 0.89 2.4 4.9 10.4 20.8 2.9 12.3 22.8 1.4 5.4 12.5 1.5 7.6 15.9 G1G0/G1 60.8 63.6 61.4 37.4 37.2 42.1 59.4 41.6 35.5 58.7 43.8 40.7 56.844.9 41.6 S 27.7 25.6 26.9 27.8 30.9 30.5 24.3 29.0 36.5 28.2 28.7 29.631.5 30.5 32.2 G2/M 11.4 10.7 11.5 34.7 31.9 27.4 16.3 29.4 28.0 13.127.5 29.7 13.5 24.5 26.1

Example 17 Isoselenocyanates are Effective for Inhibiting Melanoma TumorDevelopment in Preexisting Tumors in Nude Mice

Tumor kinetics are measured by subcutaneous injection of 2.5−5×10⁶ 1205Lu or UACC 903 melanoma cells in 0.2 ml of DMEM supplemented with 10%FBS above both left and right rib cages of 4-6 week old female nude mice(Harlan Sprague Dawley, Indianapolis, Ind.). Six days later when a fullyvascularized tumor has formed, mice are randomly divided into DMSOvehicle control and experimental groups including 5 mice/group, eachmouse having two tumors. Mice are treated by intraperitoneal (i.p.)injection with an ITC compound BITC, PEITC, PBITC, or PHITC (2.5 μmolesor 0.76 mmoles); or treated by i.p injection with an ISC compoundISC-1-ISC-2, ISC-4 or ISC-6 (0.76 mmoles, equivalent to 3 ppm selenium)thrice per week. (Monday, Wednesday and Friday). Control mice receivedan equal volume of the vehicle. Dimensions of the developing tumors aremeasured using calipers and the size estimated in cubic millimeters.Body weight is monitored three times a week (Monday, Wednesday andFriday).

FIGS. 5A and 5B show graphs comparing the effect of isothiocyanates andisoselenocyanates on tumor development using UACC 903 melanoma cells, acell line having high Akt3 signaling activity. Six days followingsubcutaneous injection of 5 million UACC 903 melanoma cells, smallvascularized palpable tumors are seen and mice are treated i. p. with2.5 μmoles of an isothiocyanate or with 0.76 μmoles, equivalent to 3 ppmselenium, of an isoselenocyanate thrice per week. The bar graphs inFIGS. 5A and 5B show melanoma tumor volume, as % of vehicle, at day 24.Isoselenocyanates ISC-2, ISC-4 and ISC-6 reduce tumor volume at doses 3×lower than isothiocyanates. ISC, 0.76 μmoles vs. ITC, 2.5 μmoles. Thus,isoselenocyanates have increased in vivo tumor inhibitory effectivenesscompared to corresponding isothiocyanates and effectively reducemelanoma development. Increasing carbon chain length of isothiocyanatesshowed less effective tumor inhibition, FIG. 5A, while increasing carbonchain length of isoselenocyanates led to greater tumor inhibition, FIG.5B.

Example 18 Isoselenocyanates Retard Melanoma Tumor Development

The effect of isothiocyanates and isoselenocyanates on tumor developmentis measured in existing tumors formed by subcutaneous injection of 2.5or 5 million 1205 Lu or UACC 903 melanoma cells. Six days followinginjection of the cells, when small vascularized palpable tumors areseen, mice are treated i. p. with PBITC or PHITC (0.76 μmoles) or ISC-4or ISC-6 (0.76 μmoles, equivalent to 3 ppm selenium) thrice per week.

FIGS. 6A and 6B show graphs of change in tumor size and body weight overtime in mice treated i. p. with PBITC or PHITC (0.76 μmoles) or ISC-4 orISC-6 (0.76 μmoles, equivalent to 3 ppm selenium) thrice per week.Selenium containing analogs ISC-4 or ISC-6 significantly reduced tumordevelopment compared to DMSO, PBITC or PHITC controls. While PBITC andPHITC are ineffective at reducing tumor burden of UACC 903, FIG. 6A, or1205 Lu, FIG. 6B, at the administered concentration of 0.76 μmoles,administration of 0.76 μmoles of ISC-4 or ISC-6 led to significantreductions of 50-60% in tumor size of both cell types. No obvioustoxicity is observed by significant changes in body weight. Data arepresented as mean±SE. Thus, isoselenocyanates (ISC) are effective atreducing melanoma tumor development at significantly lowerconcentrations than corresponding isothiocyanates (ITC).

Example 19 Measurement of Proliferation/Apoptosis Rates in Tumors

Tumors are established by subcutaneous injection of 2.5-5×10⁶ 1205 Lu orUACC 903 melanoma cells in 0.2 ml of DMEM supplemented with 10% FBSabove both left and right rib cages of 4-6 week old female nude mice(Harlan Sprague Dawley, Indianapolis, Ind.). Six days later when a fullyvascularized tumor forms, mice are randomly divided in to DMSO vehiclecontrol and experimental (BITC, PEITC, PBITC, PHITC, ISC-1-ISC-2, ISC-4or ISC-6) groups (5 mice/group; 2 tumors/mouse) and treated i. p. withITC compounds (2.5 moles or 0.76 moles), ISC compounds (0.76 moles,equivalent to 3 ppm selenium) thrice per week. (Monday, Wednesday andFriday). Control mice receive an equal volume of the vehicle. Dimensionsof the developing tumors are measured using calipers and the sizeestimated in cubic millimeters. Body weight is monitored three times aweek (Monday, Wednesday and Friday).

Isoselenocyanates decrease the tumorigenic potential of melanoma cellsby increasing apoptosis.

FIG. 7A shows that ISC treatment leads to apoptosis in size and timematched melanoma tumors. Mice bearing tumors matched for size and timeof development are injected i. p. with PBITC (0.76 moles), ISC-4 (3 ppmequivalent to 0.76 moles) or DMSO (50 μl) vehicle starting 6 days aftersubcutaneous injection of cells and on alternate days thereafter up today 15. Following ISC-4 treatment, rates of tumor cell apoptosis andcell proliferation are compared in size and time matched tumors fromISC-4 or PBITC treated animals compared to DMSO vehicle. Tumors areremoved from euthanized mice on days 9, 11, 13, and 15 for measurementof apoptosis and proliferation. Apoptosis and cell proliferation aremeasured in formalin-fixed, paraffin-embedded tumor sections using theTUNEL TMR Red Apoptosis kit from Roche (Manheim, Germany) or purifiedmouse anti-human Ki-67 from PharMingen (San Diego, Calif.),respectively.

Tumors harvested at day 11 and 13 from mice treated with ISC-4 showed˜3-fold (p<0.01; One-way ANOVA) more TUNEL positive cells compared tocontrol animals treated with DMSO or PBITC, as shown in FIG. 7A. Incontrast, slightly fewer proliferating tumor cells are observed in ISC-4treated tumors compared to PBITC but this difference is notstatistically significant (p>0.05; One-way ANOVA), shown in FIG. 7B.

A 3-fold increase in number of apoptotic cells is observed followingtreatment of UACC 903 tumors with ISC-4 at Day 11 and 13 respectivelycompared to PBITC or DMSO control. No significant changes are observedin rate of proliferation. Values shown in FIGS. 7A and 7B are means from2 separate experiments with 4-6 fields analyzed from each of 6 tumorsper experiment; bars, ±SEM. ISC-4 is more potent than PBITC ininhibiting melanoma tumor development by increasing apoptosis levels inmelanoma tumors.

Example 20 Toxicity Assessments

4-6 weeks old female nude mice (Harlan Sprague Dawley, Indianapolis,Ind.) are injected i. p. with either control DMSO vehicle, PBITC orPHITC (0.76 μmoles) or ISC-4 or ISC-6 (0.76 μmoles equivalent to 3 ppmSe) (n=5), 3 times per week (Monday, Wednesday and Friday) for 3 weeks.Animals are sacrificed by CO₂ asphyxiation and blood collected from eachanimal in plasma separator tubes with lithium heparin (BD Microtainer,BD, Franklin Lakes, N.J.) following cardiac puncture and analyzed forAST (aspartate aminotransferase), ALT, (alanyl aminotransferase),alkaline phosphatase, glucose and creatinine to ascertain liver, heart,kidney and pancreas related toxicity. For morphological examination ofblood cells, whole blood is collected in microtainer tubes containingK₂EDTA (BD Microtainer, BD, Franklin Lakes, N.J.) and RBC, WBC,lymphocytes, monocytes, eosinophils, platelets, total hemoglobin andhematocrit percentage analyzed. Blood is also microscopically examinedfor segregates, polychromatin bodies, and smudge cells. A portion ofliver, heart, kidney, spleen, intestine pancreas and adrenal from eachanimal is formalin fixed and paraffin-embedded to examinetoxicity-related changes in cell or organ morphology by H&E staining.

Body weights of isothiocyanate or isoselenocyanate treated mice comparedto the control DMSO vehicle exposed mice showed no significantdifferences between groups as shown in the two graph inserts in FIGS. 6Aand 6B.

Levels of SGOT, SGPT, alkaline phosphatase, glucose and creatinine areanalyzed in the blood collected from the animals treated with PBITC,ISC-4 or DMSO vehicle. FIG. 8 shows a bar graph indicating thatsynthetic isoselenocyanates have negligible toxicity and treatment withPBITC or ISC-4 did not alter parameters compared to vehicle DMSO treatedanimals indicating manageable toxicity to vital organs with theseagents.

Furthermore, blood parameters (SGOT, SGPT, alkaline phosphatase, bloodurea, glucose and creatinine) indicative of systemic toxicity did notdetect significant liver, kidney or cardiac related toxicity. Levels ofcellular metabolites basal urea nitrogen (BUN), creative and glucose inanimals are also not significantly different between ISC-4 or PBITCtreated and control animals. Histological examination of hematoxylin andeosin stained vital organ sections, including the liver reveal thatISC-4 treatment did not significantly change cell morphology or organstructure. Thus, synthetic selenium containing analog isothiocyanatetreatment had negligible associated systemic toxicity at theconcentrations examined with significant therapeutic potential.Synthetic isoselenocyanate compounds cause negligible organ relatedtoxicity following systemic administration.

Example 21 Statistical Analysis

Statistical analysis is undertaken using the One-way or Two-way ANOVAfollowed by the Tukey's or Bonferroni's post hoc tests. Results areconsidered significant at a p-value of <0.05.

Example 22 Western Blot Analysis of Melanoma Cells Treated withIsoselenocyanates ISC-4 and ISC-6

For Western blot analysis, floating and attached melanoma cells treatedwith compounds or control vehicle are harvested by addition of lysesbuffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 10 mM EDTA, 10%glycerol, 1% Triton X-100, 1 mM sodium orthovanadate, 0.1 mM sodiummolybdate, 1 mM phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, and 5μg/ml leupeptin. Whole cell lysates are centrifuged (≧10,000×g) for 10minutes at 4° C. to remove cell debris. Protein concentrations arequantitated using the BCA assay from Pierce (Rockford, Ill.), and 30 μgof lysate loaded per lane onto NuPAGE Gels from Life Technologies(Carlsbad, Calif.). Following electrophoresis, samples are transferredto polyvinylidene difluoride membrane (Pall Corporation, Pensacola,Fla.). Blots are probed with antibodies according to each supplier'srecommendations: phosphorylated PRAS40 (Thr246) from Invitrogen(Carlsbad, Calif.); Erk2, α-enolase and secondary antibodies conjugatedwith horseradish peroxidase from Santa Cruz Biotechnology (Santa Cruz,Calif.); and antibodies to Akt3, phosphorylated-Akt (Ser473),phosphorylated-Erk 1/2 (Thr202/Tyr204) and cleaved PARP from CellSignaling Technology (Danvers, Mass.). Immunoblots are developed usingthe enhanced chemiluminescence detection system (Pierce Biotechnology,Rockford, Ill.).

Melanoma cells (UACC 903, 1205 Lu or WM 115) are exposed to DMSO orincreasing concentrations (2.5-15 μM) of phenylbutylisothiocyanate(PBITC), phenylhexylisothiocyanate (PHITC), phenylbutylisoselenocyanate(ISC-4) or phenylhexylisoselenocyanate (ISC-6) for 24 hours. FIG. 9shows a representative Western blot analysis for expression/activity ofthe Akt signaling pathway and demonstrates dose dependent decrease inphosphorylated (active) Akt (S473) and downstream PRAS40 (T246) and acorresponding dose dependent increase in cleaved PARP, reflective ofhigh levels of cellular apoptosis. A higher level of apoptosis isobserved in cells treated with ISC-4 than in cells treated with PBITC.Erk2 served as control for equal protein loading. Isoselenocyanatesdecrease Akt3 signaling in cultured melanoma cells and tumors.

The Western blot in FIG. 9 shows that the isoselenocyanate compoundsISC-4 and ISC-6 are effective at lower concentrations compared to theisothiocyanate compounds, completely inhibiting the pathway at 10 μMcompared to corresponding isothiocyanates requiring ≧15 μM for similarinhibition. Akt3 pathway inhibition led to significant apoptosis asmeasured by the high levels of cleaved PARP. Higher levels of cleavedPARP are observed at lower concentrations of ISC-4 and ISC-6 thancorresponding isothiocyanates PBITC or PHITC.

FIGS. 10A and 10B illustrate Western blots showing the effect of ISC-4on Akt signaling pathway in melanoma cell lines 1205 Lu and WM115,respectively. ISC-4 causes significant apoptosis indicated by elevatedcleaved PARP protein levels. Erk-2 served as a control for equal proteinloading.

Thus, isoselenocyanates ISC-4 and ISC-6 more effectively inhibit theAkt3 signaling cascade in cultured melanoma cells at lowerconcentrations than corresponding sulfur containing isothiocyanates.

Example 23 Western Blot Analysis of Excised Tumors

To ascertain the mechanism underlying tumor inhibition, 5×10⁶ UACC 903cells are injected into nude mice, 6-days later mice are treated i. p.with PBITC (0.76 μmoles) or PHITC (0.76 μmoles), ISC-4 (0.76 μmoles,equivalent to 3 ppm selenium) or ISC-6 (0.76 μmoles, equivalent to 3 ppmselenium) on alternate days. Size and time matched tumors are harvestedat days 9, 11 and 13 and a small portion of the tumor is flash frozen inliquid nitrogen, pulverized and lysed in protein lysis buffer (600-80050 mM Tris-HCl, pH 7.5 containing 0.1% Triton X-100, 1 mM EDTA, 1 mMEGTA, 50 mM sodium fluoride, 10 mM sodium β-glycerol phosphate, 5 mMsodium pyrophosphate, 1 mM activated sodium orthovanadate, proteaseinhibitor cocktail from Sigma and 0.1% (v/v) 2-mercaptoethanol). Proteinconcentration is determined using Bio-Rad protein assay reagent (Bio-Radlaboratories, Hercules, Calif.) and analyzed by Western blotting tomeasure levels of pAkt and downstream pPRAS40 in tumors.

Western blot analysis of tumors harvested at day 13 from animals treatedwith DMSO, PBIT or ISC-4 showed significantly decreased expression ofphosphorylated (active) Akt (p<0.05; One-way ANOVA) and downstreamPRAS40 (p<0.001; One-way ANOVA) in ISC-4 tumor lysates compared to DMSOcontrol or PBITC treated tumors. FIG. 11 illustrates a graph ofquantitation of Western blot analysis of tumor protein lysates fromanimals treated with DMSO, PBITC or ISC-4 and indicates decreasedrelative expression of phosphorylated (active) Akt and downstream PRAS40of Akt3. Thus, isoselenocyanates ISC-4 and ISC-6 more effectivelyinhibit the Akt3 signaling cascade in tumors at lower concentrationsthan corresponding sulfur containing isothiocyanates.

Example 24

Effects of inhibiting Akt3 signaling on melanoma tumorigenesis are shownusing siRNA to inhibit Akt3 protein expression and thereby inhibit Akt3activity.

Duplexed “Stealth” siRNA from Invitrogen (Carlsbad, Calif.) are:AKT3—GGA CUA UCU ACA UUC CGG AAA GAU U (SEQ ID NO. 1) and scrambled—AAUUCU CCG AAC GUG UCA CGU GAG A (SEQ ID NO. 2). Nucleofection using AmaxaNucleofector (Koeln, Germany) is used to introduce siRNA into UACC 903cells (Reagent R, program K17). SiRNA (100 pmoles) against Akt3 orscrambled siRNA are nucleofected into 1×10⁶ UACC 903 cells, which arethen replated in DMEM supplemented with 10% FBS and allowed to recoverfor 1.5 days. Thirty-six hours later 1×10⁶ UACC 903 cells in 0.2 ml ofDMEM supplemented with 10% FBS are injected subcutaneously into the leftand right flanks of 4 to 6 week old nude mice. Control cells arenucleofected with scrambled siRNA or nucleofection buffer only.Dimensions of developing tumors are measured on alternate days usingcalipers up to day 17.5. siRNA-mediated inhibition of Akt3 signalingreduced the tumorigenic potential of melanoma cells.

FIG. 12 is a graph showing that decreased expression (activity) of Akt3reduced the tumor size in animals injected with Akt3 siRNA treated cellscompared to control cells nucleofected with scrambled siRNA ornucleofection buffer. The tumorigenic potential of melanoma cells isdecreased by ˜60%. Thus, inhibition of Akt3 signaling led to significantmelanoma tumor inhibition.

FIG. 13 is a Western blot analysis of tumor protein lysates showingreduction in expression of Akt3, demonstrating effective knockdown ofthese proteins in tumors by siRNA directed against Akt3. α-enolaseserved as loading control. A tumor removed from animals 8 days afterintroduction of siRNA shows significantly less Akt3 protein than controltumors into which a scrambled siRNA had been introduced, demonstratingeffective knockdown of Akt3 protein expression using this approach.

Example 25

Five million UACC903 cells are injected subcutaneously in a volume of200 uL at each of two sites in nude mice to establish tumors. Six daysfollowing injection of the cells, mice are divided into four groups offive mice each. Mice in each group are injected i.p with 0.76 micromolesof sulforaphane (SFN), 0.76 micromoles (equivalent to 3 ppm selenium) of1-isothiocyanto-4-methylselenobutane (SFN Iso Se Me), 0.76 micromoles(equivalent to 3 ppm selenium) of1-isoselenocyanto-4-methylsulfinylbutane (SFN Iso Se) or DMSO (a vehiclecontrol).

FIG. 14A shows inhibition of tumor development by1-isoselenocyanto-4-methylsulfinylbutane (SFN Iso Se) compared tocontrols. No toxicity of the administered compounds is apparent asindicated by lack of significant changes in body weight, shown in FIG.14B.

IC₅₀ values for sulforaphane and1-isoselenocyanto-4-methylsulfinylbutane (SFN Iso Se) are established invarious cell lines. Results are shown in Table V.

TABLE V IC-50 values Cell type SFN SFN Iso Se A549 (lung) 73 ± 3.6   35± 1.7 UACC-903 (Melanoma) 40 ± 0.89   17 ± 0.95 1205LU (Melanoma) >5039.2 ± 2   CaCO2 (Colon Carcinoma) 48 ± 5.9   16 ± 0.4 MDA-MB 231(breast) 57 ± 5     20 ± 0.46 PC-3 (Prostate) >50 24.8 ± 0.58 HT1080(Soft Tissue Sarcoma) >50 25.59 ± 1.82  IGROV-1 (ovariancarcinoma >50 >50 FF2441 (fibroblast cells) >50 >50

Example 26

Effect of ISC and ITC compounds on cell viability of prostate cancercells LNCaP, PC-3 and DU-145.

Prostate cell lines, LnCaP, PC-3 and DU-145 are exposed to differentconcentrations, 5 micromolar, 10 micromolar or 25 micromolar, of ITCsincluding BITC, PEITC, PBITC, and PHITC, or ISCs, including ISC-1,ISC-2, ISC-4, and ISC-4, for 24 hours. Following incubation, cellviability is assayed using an MTS assay.

Example 27

In vitro inhibitory efficacy of DMSO, ISC-1, ISC-2, ISC-4, ISC-6, BITC,PEITC, PBITC, or PHITC on UACC 903 cells is measured using the3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) assay (Promega, Madison, Wis.). In brief, 2.5-5×103 cells per wellin 100 microliters DMEM containing 10% FBS are grown in a 96-well platefor 24 h and treated with either control DMSO vehicle or increasingconcentrations (5-15 μM) of the tested isothiocyanate orisoselenocyanate for 24 hours. At this point cells are treatedindividually with either vehicle control DMSO or with increasingconcentrations of ITC or ISC (2.5-100 μM) for 24 h. The percentages ofviable cells compared to control DMSO treated cells are determined. FIG.15 shows results of this assay.

Example 28

Cellular proliferation rate is measured by seeding 5×10³ human melanomacell line UACC 903 cells in a 96-well plate, followed by treatment for24 hours with DMSO or 5-15 μM of ISC-1, ISC-2, ISC-4, ISC-6, BITC,PEITC, PBITC, or PHITC for 24 hours. Proliferation rates are measuredusing a BrdU ELISA kit (Roche Applied Sciences, Indianapolis, Ind.).Results of this assay are shown in FIG. 16.

Example 29

Apoptosis rate is measured by seeding 5×10³ human melanoma cell lineUACC 903 cells in a 96-well plate, followed by treatment for 24 hourswith DMSO or 5-15 μM of ISC-1, ISC-2, ISC-4, ISC-6, BITC, PEITC, PBITC,or PHITC for 24 hours. Apoptosis rate is measured using an Apo-ONEHomogenous caspase-3/7 Assay kit (Promega Corporation, Madison, Wis.).Results of this assay are shown in FIG. 17.

Example 30 Tumorigenicity Assessment

Tumor kinetics are measured by subcutaneous injection of 5×10⁶ UACC 903melanoma cells in 0.2 ml of DMEM supplemented with 10% FBS above bothleft and right rib cages of 4-6 week old female nude mice (HarlanSprague Dawley, Indianapolis, Ind.). Six days later mice are randomlydivided into control (DMSO) and experimental (ISC-1, ISC-2, ISC-4,ISC-6, BITC, PEITC, PBITC, PHITC) groups (5 mice/group; 2 tumors/mouse).Six days after subcutaneous injection of UACC 903 melanoma cells, miceare treated i. p. with BITC, PEITC, PBITC or PHITC (2.5 μmoles), or,ISC-1, ISC-2, ISC-4 or ISC-6 (0.76 μmoles, equivalent to 3 ppm selenium)thrice per week. (Monday, Wednesday and Friday). Control mice receive anequal volume of the vehicle, DMSO. The dimensions of the developingtumors (using calipers) and body weight are measured three times a week(Monday, Wednesday and Friday) and the size estimated in cubicmillimeters.

At a dose of 0.76 μmoles, ISC-2 to ISC-6 showed about 30-45% reductionin tumor size with the effect increasing with the increasing alkyl chainlength from ISC-2 to ISC-6 (FIG. 18A). ISC-1 failed to show any effectat this concentration. There was no related toxicity observed at thisdose for any of the ISC derivatives. ITC derivatives are also effectivein reducing the tumor size at a dosage of 2.5 micromoles, ˜3 timeshigher than the administered dosage of ISC compounds, shown in FIG. 18C.A reverse trend of chain length effect is observed, with BITC being themost effective. FIGS. 18B and 18D shows the body weights of testcompound treated mice treated compared to the control DMSO vehicletreated mice. No significant difference in weights is detected betweengroups, demonstrating negligible toxicity.

Example 31 Western Blotting, Immunoprecipitation, and Kinase Assay forAkt1, Akt2 and Akt3

Dysregulation of Akt1, Akt2 and/or Akt3 can be observed in cells usingvarious well-known techniques

Western blot procedure for detection of Akt1, Akt2 and/or Akt3dysregulation is performed according to standard procedures, forinstance as described herein and in Harlow, E. and Lane, D., Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988; J. D.Pound (Ed.) Immunochemical Protocols, Methods in Molecular Biology,Humana Press; 2nd ed., 1998; Stahl, J. M., et al., Cancer Res.,63:2881-2890, 2003; and Stahl, J. M., et al., Cancer Res., 64:7002-7010,2004.

Immunoprecipitation for detection of Akt1, Akt2 and/or Akt3dysregulation is performed according to standard procedures, forinstance as described herein and in Harlow, E. and Lane, D., Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988; J. D.Pound (Ed.) Immunochemical Protocols, Methods in Molecular Biology,Humana Press; 2nd ed., 1998; Stahl, J. M., et al., Cancer Res.,63:2881-2890, 2003; and Stahl, J. M., et al., Cancer Res., 64:7002-7010,2004. For example, briefly described, protein is collected from cellsafter addition of protein lysis buffer [50 mmol/L Tris-HCl (pH 7.5),0.1% Triton X-100, 1 mmol/L EDTA, 1 mmol/L EGTA, 50 mmol/L NaFl, 10mmol/L sodium β-glycerol phosphate, 5 mmol/L sodium inorganicpyrophosphate, 1 mmol/L sodium orthovanadate, 0.1% 2-mercaptoethanol,and 0.5% protease inhibitor mixture (Sigma, St. Louis, Mo.)] to asample, such as plates of cells or biopsy material, followed by snapfreezing in liquid nitrogen. Cellular debris is pelleted bycentrifugation (10,000×g) of lysates, and protein concentration isquantitated using the Bio-Rad Protein Assay (Bio-Rad, Hercules, Calif.).Protein for immunoprecipitation (100 μg) is incubated with 2 μg of Akt1or Akt2 or 5 μL of Akt3 antibody overnight at 4° C. with constantmixing. A no antigen negative control is prepared by adding antibody tothe lysis buffer only. Next, 15 μL of equilibrated GammaBind G Sepharosebeads (Amersham Biosciences, Piscataway, N.J.) is added to each tube andincubated for 2 hours (4° C.) with constant mixing. Pelleted beads arewashed twice with lysis buffer to remove unbound antibody and protein.Samples are then electrophoresed under reducing conditions according tothe protocol provided by Invitrogen Life Technologies, Inc. (Carlsbad,Calif.) with the NuPage Gel System. Western blots of the electrophoresedsamples are probed with an anti-phospho-Akt (Ser-473) antibody andquantitated by densitometry as described in Stahl, J. M., et al., CancerRes., 63:2881-2890, 2003.

Antibodies for use in immunoassays for Akt1, Akt2 and/or Akt3 can beobtained commercially, for instance an Akt1 antibody is available fromCell Signaling Technologies, Beverly, Mass., an Akt2 antibody isavailable from Santa Cruz Biotechnology, Santa Cruz, Calif. and an Akt3antibody is available from Upstate Biotechnology, Lake Placid, N.Y. Ananti-phospho-Akt (Ser-473) antibodies can also be obtained commercially,e.g. from Cell Signaling Technologies, Beverly, Mass. Anti-Akt1, Akt2and/or Akt3 antibodies can also be produced by well-known techniquessuch as described, for instance, in Harlow, E. and Lane, D., Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988.

Akt kinase assays are performed according to standard kinase assayprocedures. Briefly described, 15 μL of equilibrated GammaBind GSepharose beads are incubated with 2 μg of Akt1 or Akt2 antibody, or 5μL of Akt3 antibody in a volume of 350 μL of lysis buffer at 4° C. withconstant mixing for 2 hours. Microcystin (1 μmol/L) from MP Biomedicals(Irvine, Calif.) is added to the lysis buffer to ensure completeinactivation of cellular PP1 and PP2 phosphatases. Theantibody/Sepharose complex is washed twice with 750 μL of lysis bufferand then incubated with 100 μg of protein in a volume of 350 μLovernight at 4° C. with constant mixing. This complex is washed with 500μL, of lysis buffer (3×) and then once with 500 μL of assay dilutionbuffer [20 mmol/L 4-morpholinepropanesulfonic acid (pH 7.2), 25 mmol/Lβ-glycerol phosphate, 1 mmol/L sodium orthovanadate, and 1 mmol/Ldithiothreitol]. Protein kinase A (PKA) inhibitor peptide (10 μmol/L)from Santa Cruz Biotechnology (Santa Cruz, Calif.), 37.5 mon ATP, 17mmol/L MgCl₂, 0.25 μCi/μL [gamma-³²P]ATP, and 90 μmol/L, Akt-specificsubstrate Crosstide from Upstate Biotechnology (Lake Placid, N.Y.) areadded to the tubes in assay dilution buffer and incubated at 35° C. for10 minutes with continuous mixing. Next, 20 μL of liquid are transferredto phosphocellulose paper, which is washed three times for 5 minuteswith 40 mL of 0.75% phosphoric acid. After a 5-minute acetone wash, thephosphocellulose was allowed to dry and transferred to a scintillationvial with 5 mL of Amersham Biosciences scintillation fluid, and countsper minute were measured in a Beckman Coulter LS 3801 LiquidScintillation System (Fullerton, Calif.).

Detection of Dysregulated Akt in Human Material

Formalin-fixed paraffin-embedded melanoma specimens are used forimmunohistochemistry to measure phosphorylated Akt. A phospho-Akt(Ser-473) monoclonal antibody, Cell Signaling Technologies, is used at a1:50 titer according to the manufacturer's recommended protocol.Specificity and intensity of staining are determined through qualitativecomparison with internal blood vessel endothelium, squamous epithelium,or smooth muscle controls present in each specimen.

Example 32

ISC-4 more efficiently inhibits melanoma cell growth compared to normalhuman fibroblast cells. 10×10³ normal human fibroblasts (FF2441)expressing Akt3 at normal levels and 5×10³ metastatic melanoma cells(UACC 903) having activated Akt3 are plated in 96 well plates in 100 μLDMEM containing 10% FBS and grown for 24 hours respectively.Exponentially growing cells are treated with increasing concentrations(2.5-100 μM) of ISC-4 for 24 hours and IC₅₀ (μM) values determined.

Sensitivity of melanoma cells to ISC-4 with elevated Akt3 signaling iscompared to fibroblast cells with normal levels of Akt activity.Three-fold higher drug concentration of ISC-4 (37.5 μM) is required tokill fibroblasts with normal levels of Akt activity compared to melanomacells (12±3 μM) with elevated Akt activity. Thus, cancer cells withconstitutively active Akt signaling are 3-fold more sensitive to ISC-4than normal cells with regular Akt activity. These results show thatISC-4 more effectively kill cells having increased Akt activity thanthose having normal levels.

Example 33

Effect of topical ISC-4 application on melanoma tumor growth—in vitro(Skin Reconstructs). FIG. 19 is a bar graph showing the effects oftopically applied PBITC or ISC-4 on reconstructed human skin containingGFP tagged UACC 903 human melanoma cells.

Generation of skin containing melanocytic lesions is briefly described.To create skin in a culture dish, human fibroblasts, were trypsinizedand resuspended in 10% reconstitution buffer, 10% 10×DMEM (Mediatech,Herndon, Va.), 2.4 microliters/ml of 10 M NaOH, and 80% collagen I (BDDiscovery Labware Inc., Bedford, Mass.) at a concentration of 2.5×10⁵cells/ml on ice (Ozbun M A, Meyers C. J Virol 1996, 70: 5437-46).Mixture was then aliquoted into 6 or 12 well plates and incubated at 37°C. for 3 hours. E-media was added to each well to equilibrate the dermalmatrix (Wu Y J, Parker L M, Binder N E, et al., Cell 1982, 31: 693-703).After two days of growth, keratinocytes and melanoma cells (WM35-GFP orUACC 903-GFP) were trypsinized and resuspended at a 1:10 ratio ofmelanoma cells (nucleofected or untreated) to keratinocytes in E-media.One milliliter of cell suspension added to each well on top of thedermal layer. Following two days growth, reconstructed skin wastransferred onto wire grids and fed via diffusion from E-media below theplatforms.

Reconstructed human skin containing GFP tagged UACC 903 human melanomacells were treated with 12.5 and 25 μM PBITC or ISC-4 and the tumor areaoccupied measured using fluorescence microscopy.

The result shows an ˜80% decrease in tumor area occupied by melanomacells upon ISC-4 treatment compared to control DMSO treated or untreatedskins. Similar results are observed with other melanoma cell lines.

Example 34 Effect of Topical ISC-4 Application on Melanoma TumorGrowth—In Vivo

Chemopreventive or chemotherapeutic effect of ISC-4 on cutaneous tumordevelopment is measured by subcutaneous injection of 1 million UACC 903cells in 0.2 ml of DMEM −10% FBS above both the left and right rib cagesof 4- to 6-week old female athymic nude mice using 24 g needles. 24hours later, animals are treated daily with ISC-4 (0.063-0.19 mmolesequivalent to 0.25-0.75 ppm), PBITC (0.063-0.19 mmoles) or vehiclecontrol (acetone) for 3-4 weeks. The dimensions of the developing tumorsare measured alternate days using calipers and the sizes estimated incubic millimeters. A minimum of 5 mice per group is used for the topicaltreatment.

FIG. 20 shows a pair of line graphs showing the effect of topical ISC-4application on melanoma tumor growth in vivo. Topical treatment with(0.063-0.19 μmoles equivalent to 0.25-0.75 ppm) ISC-4 leads to decreasedtumor size compared to vehicle control (as shown in the upper graph inFIG. 20) or PBITC (0.063-0.19 μmoles) with no systemic toxicity (asshown by body weight measurements in the lower graph in FIG. 20).

Example 35 Effect of Topical ISC-4 Application on Melanoma TumorGrowth—In Vivo

Chemopreventive or chemotherapeutic effect of ISC-4 on cutaneous tumordevelopment is measured by subcutaneous injection of 1 million UACC 903cells in 0.2 ml of DMEM −10% FBS above both the left and right rib cagesof 4- to 6-week old female athymic nude mice using 24 g needles. 24hours later, animals were treated daily with ISC-4 (12.5-50 μM), PBITC(12.5-50 μM), or vehicle control (acetone) for 3-4 weeks. The dimensionsof the developing tumors are measured alternate days using calipers andthe sizes estimated in cubic millimeters. A minimum of 5 mice per groupwas used for the topical treatment.

Topical treatment with ISC-4 (12.5-50 μM) significantly reduces melanomatumor development by ˜50% compared to PBITC (12.5-50 μM) (as shown inthe upper graph in FIG. 21) or vehicle control with no systemic toxicity(as shown by body weight measurements in the lower graph in FIG. 21).

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Any patents or publications mentioned in this specification areincorporated herein by reference to the same extent as if eachindividual publication is specifically and individually indicated to beincorporated by reference. Pending U.S. patent application Ser. No.12/102,629 and U.S. Provisional Patent Application Ser. Nos. 60/911,565,filed Apr. 13, 2007 and 60/959,554, filed Jul. 13, 2007, are allincorporated herein by reference in their entirety.

The compositions and methods described herein are presentlyrepresentative of preferred embodiments, exemplary, and not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art. Such changes and other usescan be made without departing from the scope of the invention as setforth in the claims.

1. A composition comprising an isoselenocyanate having the structuralformula: R—(CH₂)_(n)—N═C═Se, where n is an integer in the range of 1-8,inclusive, and where R is selected from the group consisting of: anaromatic group and a non-aromatic organic group.
 2. The composition ofclaim 1 where the isoselenocyanate has the structural formula:

where R′ is a substituted or unsubstituted, branched or straight chain,lower alkyl group, and where n is an integer in the range of 3-8,inclusive.
 3. The composition of claim 2 wherein R′ is CH₃.
 4. Thecomposition of claim 2, where R′ is CH₃ and n is
 4. 5. A method forsynthesis of an isoselenocyanate having the structural formula:R—(CH₂)_(n)—N═C═Se, where n is an integer in the range of 1-8,inclusive, and where R is selected from the group consisting of: anunsubstituted aromatic group; an aromatic group substituted by one ormore substituents selected from the group consisting of: F, Cl, Br, alower alkyl group, a lower alkoxy group and a fluorinated lower alkylgroup; R′—S(O), where R′ is a lower alkyl group, which may besubstituted or unsubstituted, branched or straight chain; and CH₂═CH,comprising: formylation of a starting alkylamine compound having thestructural formula: R—(CH₂)_(n)—NH₂, where n is an integer in the rangeof 1-8, inclusive, where R is selected from the group consisting of: anunsubstituted aromatic group; an aromatic group substituted by one ormore substituents selected from the group consisting of: F, Cl, Br, alower alkyl group, a lower alkoxy group and a fluorinated lower alkylgroup; R′—S(O), where R′ is a lower alkyl group, which may besubstituted or unsubstituted, branched or straight chain, to produce aformylated intermediate having the structural formula:R—(CH₂)_(n)—NHCHO, where n and R are identical to R and n of thestarting alkylamine; contacting the formylated intermediate withtriphosgene and selenium powder in the presence of triethylamine.
 6. Themethod of claim 5, wherein the formylated intermediate is contacted withtriphosgene and selenium powder in the presence of triethylamine and inthe presence of a solvent.
 7. A pharmaceutical composition, comprising:a compound having the structural formula: R—(CH₂)_(n)—N═C═X, where n isan integer in the range of 1-8, inclusive, where X is S or Se, and whereR is selected from the group consisting of: an aromatic group and anon-aromatic organic group.
 8. The pharmaceutical composition of claim7, wherein the compound is selected from the group consisting of: aphenylalkyl isothiocyanate, a phenylalkyl isoselenocyanate, and acombination thereof.
 9. The pharmaceutical composition of claim 7,wherein the compound is an isoselenocyanate having the structuralformula selected from the group consisting of:

where n is 4 or 6; and

where R′ is a substituted or unsubstituted, branched or straight chain,lower alkyl group, and where n is an integer in the range of 1-8,inclusive; and a pharmaceutically acceptable carrier.
 10. Thecomposition of claim 7, further comprising a pharmaceutically acceptablecarrier.
 11. The composition of claim 7, wherein the pharmaceuticalcomposition is formulated for topical application.
 12. A method oftreating a subject, comprising: administering a therapeuticallyeffective amount of a composition comprising a compound having thestructural formula: R—(CH₂)_(n)—N═C═X, where n is an integer in therange of 1-8, inclusive, where X is S or Se, and where R is selectedfrom the group consisting of: an aromatic group and a non-aromaticorganic group to a subject in need thereof.
 13. The method of claim 12,wherein the composition comprises a compound having the structuralformula: phenyl-(CH₂)_(n)—N═C═X, where n is an integer in the range of1-8, inclusive, where X is S or Se.
 14. The method of claim 12, whereinthe composition comprises an isoselenocyanate having the structuralformula:

where R′ is a substituted or unsubstituted, branched or straight chain,lower alkyl group, and where n is an integer in the range of 3-8,inclusive.
 15. The method of claim 14 wherein R′ is CH₃.
 16. The methodof claim 12, wherein the composition comprises a phenylalkylisoselenocyanate having the structural formula:

where n is 4 or
 6. 17. The method of claim 12 wherein theisoselenocyanate is selected from the group consisting of: anisoselenocyanate glutathione conjugate; an isoselenocyanate cysteineconjugate; and an isoselenocyanate N-acetylcysteine conjugate.
 18. Themethod of claim 12 wherein the subject is human.
 19. The method of claim12 wherein the subject has or is at risk of having cancer.
 20. Themethod of claim 19 wherein the cancer is a melanoma.
 21. The method ofclaim 12 wherein the composition is formulated for topical application.22. The method of claim 19 wherein the cancer is characterized bydysregulation of Akt.
 23. The method of claim 19 wherein the cancer ischaracterized by dysregulation of Akt3.
 24. The method of claim 19,wherein administering the therapeutically effective amount of thecomposition to a subject detectably increases apoptosis and/or decreasesproliferation of cells of the cancer.
 25. The method of claim 19,further comprising administration of an adjunct anti-cancer treatment.26. A method of modulating Akt dysregulation in a cell, comprising:contacting the cell with an effective amount of an isoselenocyanatehaving the structural formula: R—(CH₂)_(n)—N═C═Se, where n is an integerin the range of 1-8, inclusive, and where R is selected from the groupconsisting of: an aromatic group and a non-aromatic organic group. 27.The method of claim 26 wherein contacting the cell decreases a componentof an Akt signaling pathway selected from the group consisting of anAkt1 signaling pathway; an Akt2 signaling pathway; an Akt3 signalingpathway; and a combination thereof.